Hyper-Spectral Imaging and Analysis of a Sample of Matter, and Preparing a Test Solution or Suspension Therefrom

ABSTRACT

Method for hyper-spectral imaging and analysis of a sample of matter, for identifying and characterizing an object of interest therein. Preparing test solution or suspension of the sample, including adding thereto a spectral marker specific to object of interest, such that if object of interest is in test solution or suspension, object of interest becomes a hyper-spectrally active target which is hyper spectrally detectable and identifiable; adding to test solution or suspension a background reducing chemical, for reducing background interfering effects caused by presence of objects of non-interest in test solution or suspension, thereby increasing hyper spectral detectability of hyper spectrally active target in test solution or suspension; generating and collecting hyper-spectral image data and information of test solution or suspension; and, processing and analyzing thereof. Exemplary objects of interest are biological agents—bacteria ( Bacillus anthracis ), viruses, fungi, toxins, or, chemical agents—nerve agents (sarin, tabun, soman), and chemical poisons.

FIELD AND BACKGROUND OF THE INVENTION

The present invention relates to hyper-spectral imaging and analysis ofa sample of matter, and more particularly, to a method forhyper-spectral imaging and analysis of a sample of matter, foridentifying and characterizing an object of interest therein. Thepresent invention also features a method for preparing a test solutionor suspension from a sample of matter, such that the test solution orsuspension is particularly suitable for subjecting to hyper-spectralimaging and analysis. The present invention is generally applicable foron-line (e.g., real time or near-real time) or off-line hyper-spectralimaging and analysis of various different types or kinds of samples ofmatter, wherein the matter, and at least one object of interest therein,are composed or made up of organic or/and inorganic materials orsubstances, which are in a solid (e.g., particulate) phase, a liquid(e.g., solution or suspension) phase, or/and a gaseous (e.g., aerosol)phase. The present invention provides the capability of achieving the‘ultimate’ combination of the highly desirable performance parameters ofhigh accuracy, ‘and’ high precision (high reproducibility), ‘and’ highresolution, ‘and’ high sensitivity, ‘and’ at high speed (short timescale), all at the same time (i.e., simultaneously), be it duringon-line or off-line, in an optimum and highly efficient manner.

An exemplary specific application of the present invention involveson-line (real time or near-real time) or off-line hyper-spectral imagingand analysis of a sample of air (i.e., an air sample), for identifyingand characterizing an object of interest therein, wherein the object ofinterest is a (potentially hazardous) biological agent or a (potentiallyhazardous) chemical agent. In general, the object of interest (i.e.,biological agent or chemical agent) in the air sample is composed ormade up of organic or/and inorganic materials or substances, which arein a solid (e.g., particulate) phase, a liquid (e.g., solution orsuspension) phase, or/and a gaseous (e.g., aerosol) phase. Preferably,the object of interest (i.e., biological agent or chemical agent) in theair sample is composed or made up of organic or/and inorganic materialsor substances, which are in a particulate form solid phase or/and arepresent (e.g., absorbed or/and adsorbed) on particles of the air sample.The sample of air is collected or obtained (e.g., via a standard type ofair sampling or collecting system) from an indoor source of air (e.g., apost office, an airport, a subway station, a shopping mall, a sportsarena, or an office building), or from an outdoor source of air.Exemplary biological agents are bacteria, viruses, fungi, and toxins.Exemplary chemical agents are nerve agents (e.g., sarin, tabun, andsoman), and chemical poisons (e.g., cyanide compounds, andorganophosphate compounds). The object of interest can be a biologicalagent, such as the (extremely hazardous) spore-forming bacteriumBacillus anthracis, which is chemically marked (e.g., via terbiumtrichloride [TbCl₃]), or biologically marked (e.g., via antibodies of animmunoassay technique), as part of the main step (procedure) ofpreparing a test solution or suspension of the sample of matter, i.e.,the air sample, for enabling identification and characterization thereofvia hyper-spectral imaging and analysis.

Sample of Matter

A sample of matter generally refers to a relatively small quantity ofmatter which is representative of, and an example of, (i.e., a sample),of a relatively large, quantity of the matter, where matter generallyrefers to something (i.e., entity, material, substance) that has mass,occupies volume, and exists as a solid, liquid, gas, or a combinationthereof. A sample of matter may also be considered as being a specimen(i.e., example) of the matter. Herein, the term ‘object’ generallyrefers to, and is considered equivalent to, and synonymous with, atleast part of a given matter, and therefore, that which is present in asample of the matter. Accordingly, the term ‘object’ generally refersto, and is considered equivalent to, and synonymous with, at least partof something (i.e., entity, material, substance) that has mass, occupiesvolume, and exists as a solid, liquid, gas, or a combination thereof,and therefore, that which is present in a sample of matter. Moreover,each object (i.e., at least part of a given matter) is definable andcharacterizable by a set of a wide variety of numerous possiblebiological, chemical, or/and physical, properties, characteristics, andbehavior.

Analyzing a Sample of Matter

In essentially every field or area of science and technology there areapplications which are based on, or involve, the need for on-line (realtime or near-real time) or off-line analyzing a sample of matter, for amain purpose or objective of identifying and characterizing at least oneobject (i.e., entity, material, substance) of interest, usually among avariety of different types or kinds of objects (i.e., entities,materials, substances) of non-interest, in the sample of matter. Suchcharacterization may include determining any number and types or kindsof biological, chemical, or/and physical, properties, characteristics,features, parameters, or/and behavior, of the at least one object ofinterest in the sample of matter.

There exists a plethora of prior art teachings and practices of a widevariety of different analytical methods and techniques, and associatedanalytical equipment, instrumentation, hardware, and software, which aresuitable for on-line (real time, near-real time) or off-line analyzing asample of matter. Clearly, many factors, parameters, conditions,criteria, and requirements, are involved that need to be identified,analyzed, considered, accounted for, and possibly tested, in order toproperly determine which particular analytical method or technique, and,associated analytical equipment, instrumentation, hardware, andsoftware, are most suitable, alternatively suitable, or optionallysuitable, for analyzing a particular sample of matter.

Hyper-spectral imaging and analysis has been established as a highlyunique, specialized, and sophisticated, combined spectroscopy andimaging type of analytical method or technique, in the more encompassingfield or area of analytical science and technology, involving thesciences and technologies of spectroscopy and imaging. By definition,hyper-spectral imaging and analysis is based on a combination ofspectroscopy and imaging theories, principles, and practices, which areexploitable for analyzing samples of matter in a highly unique,specialized, and sophisticated, manner.

Hyper-spectral imaging and analysis, theory, principles, and practicesthereof, and, related and associated applications and subjects thereof,such as the more general subject of spectral imaging, are well known andtaught about in scientific, technical, and patent, literature, andcurrently practiced in a wide variety of numerous different fields andareas of science and technology. Several (mostly recent) examples ofsuch teachings and practices are disclosed in references 1-29 (andreferences cited therein). Selected teachings and practices ofhyper-spectral imaging and analysis by the same applicant/assignee ofthe present invention are disclosed in references 30-36. For the purposeof establishing the scope, meaning, and field(s) or area(s) ofapplication, and meaning, of the present invention, and in understandingproblems solved by the present invention, the following background isprovided.

Hyper-Spectral Imaging and Analysis

The more highly specialized, complex, and sophisticated, spectroscopicimaging technique of ‘hyper-spectral’ imaging and analysis, in contrastto the regular or standard spectroscopic imaging technique of ‘spectral’imaging and analysis, consists of using a hyper-spectral imaging andanalysis system for on-line (real time, near-real time) or off-linegenerating and collecting (acquiring) hyper-spectral images and spectra(herein, together, generally referred to as hyper-spectral image dataand information), and, processing and analyzing the acquiredhyper-spectral image data and information. In hyper-spectral imaging,multiple fields of view of a sample of matter are ‘hyper-spectrally’scanned and imaged while the sample of matter (containing objects, andcomponents thereof) is exposed to electromagnetic radiation. During thehyper-spectral scanning and imaging there is generating and collectingrelatively large numbers (up to the order of millions) of multiplespectral (i.e., hyper-spectral) images, ‘one-at-a-time’, but, in anextremely fast or rapid sequential manner, of the objects (andcomponents thereof) emitting electromagnetic radiation at a plurality ofmany wavelengths and frequencies, where the wavelengths and frequenciesare associated with different selected (relatively narrow) portions orbands, or bands therein, of an entire hyper-spectrum emitted by theobjects (and components thereof). A hyper-spectral imaging and analysissystem can be operated in an extremely fast or rapid manner forproviding exceptionally highly resolved spectral and spatial data andinformation of an imaged sample of matter, with high accuracy and highprecision (reproducibility), which are fundamentally unattainable byusing a regular or standard spectral imaging and analysis system.

In general, when electromagnetic radiation, for example, in the form oflight such as that supplied by the sun, or by a man-made imaging type ofilluminating or energy source, such as that used during hyper-spectralimaging, is incident upon an object, the electromagnetic radiation isaffected by one or more of the biological, chemical, or/and physical,species or components making up the object, by any combination ofelectromagnetic radiation absorption, diffusion, reflection,diffraction, scattering, or/and transmission, mechanisms. Moreover, anobject whose composition includes organic chemical species orcomponents, ordinarily exhibits some degree or extent of fluorescentor/and phosphorescent properties, characteristics, and behavior, whenilluminated by some type of electromagnetic radiation or light, such asultra-violet (UV), visible (VIS), or infrared (IR), types of light. Theaffected electromagnetic radiation, in the form of diffused, reflected,diffracted, scattered, or/and transmitted, electromagnetic radiationemitted by, or/and emerging from, the object, is directly and uniquelyrelated to the biological, chemical, or/and physical, properties,characteristics, and behavior, of the object, in general, and of thechemical species or components making up the object, in particular, andtherefore represents a spectral (‘fingerprint’ or ‘signature’) patterntype of identification and characterization of the object.

Accordingly, hyper-spectral images generated by, and collected from, asample of matter, are correlated with emission spectra of the sample ofmatter, where the emission spectra correspond to spectralrepresentations in the form of spectral ‘fingerprint’ or ‘signature’pattern types of identification and characterization, of thehyper-spectrally imaged objects (and components thereof) in the sampleof matter. Such hyper-spectral image data and information are processedand analyzed by using automatic pattern recognition (APR) or/and opticalcharacter recognition (OCR) types of hyper-spectral imaging data andinformation processing and analysis, for identifying, characterizing,or/and classifying, the physical, chemical, or/and biological,properties, characteristics, and behavior, of the hyper-spectrallyimaged objects (and components thereof) in the sample of matter.

Hyper-Spectral Imaging and Analysis of a Sample of Matter

Following provision of a sample of matter, or following obtaining orcollecting a sample of matter, analyzing a sample of matter viahyper-spectral imaging and analysis, similar to analyzing a sample ofmatter by essentially any analytical method or technique, involves threeseparate, but integrated, general domains or stages of main activitiesand procedures. In particular, following provision of a sample ofmatter, or following obtaining or collecting a sample of matter,hyper-spectral imaging and analysis of the sample of matter typicallyinvolves the following three separate, but integrated, general domainsor stages of main activities and procedures: (i) preparing anappropriate test form (usually, a solid or liquid form) of the sample ofmatter, which is suitable for being subjected to hyper-spectral imagingand analysis, (ii) generating and collecting hyper-spectral image dataand information of the test form of the sample of matter, and (iii)processing and analyzing the generated and collected hyper-spectralimage data and information.

In general, each of these three general domains or stages of mainactivities and procedures of a hyper-spectral imaging and analysisapplication can be characterized by various different levels or degreesof the following performance parameters: accuracy, precision(reproducibility), sensitivity, resolution, and speed. In any givehyper-spectral imaging and analysis application, the just stated threegeneral domains or stages of main activities and procedures are fullyintegrated and inter-dependent upon each other.

The scope of application of the present invention is primarily directedto, and focused on, the preceding stated first general domain or stageof main activities and procedures of a hyper-spectral imaging andanalysis application, i.e., being based on, and involving, preparing anappropriate test form of the sample of matter, which is suitable forbeing subjected to hyper-spectral imaging and analysis. However, intheory, and in practice, the performance parameters of accuracy,precision (reproducibility), sensitivity, resolution, or/and speed (timescale), of the first general domain or stage of main activities andprocedures of a hyper-spectral imaging and analysis application, i.e.,regarding preparation of an appropriate test form of the sample ofmatter, affect and influence the performance parameters of accuracy,precision (reproducibility), sensitivity, resolution, or/and speed (timescale), of each of the succeeding second and third general domains orstages of main activities and procedures. More specifically, mainactivities and procedures of preparing an appropriate test form of asample of matter, affect and influence generating and collectinghyper-spectral image data and information of the test form of the sampleof matter, which in turn, affect and influence processing and analyzingthe generated and collected hyper-spectral image data and information.Thus, the scope of application of the present invention also encompassesthe preceding stated second and third general domains or stages of mainactivities and procedures of hyper-spectral imaging and analysis of asample of matter.

Preparing an Appropriate Test Form of a Sample of Matter

This general domain or stage of main activities and procedures of ahyper-spectral imaging and analysis application involves preparing anappropriate test form of a sample of matter which is suitable for, andcompatible with, operation and use of equipment and instrumentation of agiven hyper-spectral imaging and analysis system. Typically, anappropriate test form of a sample of matter involves using a relativelysmall quantity (for example, on the order of microliters (μl)) of thesample of matter, ‘as is’, in a solid, solution, or suspension, form.Alternatively, according to the actual composition or makeup of thesample of matter (including the objects and components thereof presentin the sample of matter), an appropriate test form of a sample of mattermay involve dissolving, suspending, or/and mixing, i.e., reformulating,a relatively small quantity of the sample of matter into a solution orsuspension form. A portion or aliquot of the solid, solution, orsuspension, test form of the sample of matter is then, typically, placedon a clean, inert, metal slide or plate, or, on a clean, inert, plastic(e.g., Teflon®) or glass microscope type slide or plate, which issuitable for functioning as a sample holder in a hyper-spectral imagingand analysis system. The slide or plate (sample holder) with the portionor aliquot of the test solution or suspension of the sample of matter isthen appropriately positioned and secured (fixed) upon athree-dimensionally movable (i.e., translational), and optionally,angularly movable (i.e., rotational), examination stage or platform ofthe hyper-spectral imaging and analysis system.

For the purpose of fully understanding the hereinbelow describedsignificant on-going problems and limitations of hyper-spectral imagingand analysis of a sample of matter, immediately following is a briefdescription of hyper-spectrally imaged scenes of a test form of a sampleof matter, in terms of types, categories, or classes, of objectsexisting in the hyper-spectrally imaged scenes.

Hyper-Spectrally Imaged Scenes of a Sample of Matter, and Types,Categories, or Classes, of Objects Therein

In general, in hyper-spectrally imaged scenes of a test form of a sampleof matter (including the objects and components thereof present in thesample of matter), the objects (i.e., entities, materials, substances)can be typed, categorized, or classified, according to two maindifferent types, categories, or classes. Namely, ‘objects ofnon-interest’, and ‘objects of interest’, each of which is basicallydefined as follows. ‘Objects of non-interest’ correspond to objects of(present or contained in) a hyper-spectrally imaged scene of the sampleof matter which are not of interest to a human operator (observer,viewer, analyzer, or/and controller) of a process involving the sampleof matter. ‘Objects of interest’ correspond to objects of (present orcontained in) a hyper-spectrally imaged scene of the sample of matterwhich are of interest to a human operator of a process involving thesample of matter. For further understanding the significantly differentmeanings and attributes of objects of non-interest and objects ofinterest, in the context of the present invention, objects ofnon-interest are considered as being part of the ‘background’ of, orwithin, a hyper-spectrally imaged scene of the sample of matter, whereasobjects of interest are considered as being ‘targets’ of, or within, ahyper-spectrally imaged scene of the sample of matter. Accordingly, inhyper-spectral imaging, individual objects among a plurality,collection, or ensemble, of several objects (i.e., entities, materials,substances) of (present or contained in) a hyper-spectrally imaged sceneof a sample of matter, can be typed, categorized, or classified,according to the above stated two main different types, categories, orclasses, of objects, i.e., objects of non-interest (i.e., background),and objects of interest (i.e., targets).

Typically, each hyper-spectrally imaged scene of a sample of matterincludes or contains a distribution of different relative numbers (i.e.,ratios, proportions) of the preceding defined two main different types,categories, or classes, of objects. For example, a givenhyper-spectrally imaged scene may include or contain a distribution of arelatively small number of objects of interest (targets), and arelatively large number of objects of non-interest (corresponding to arelatively high or ‘noisy’ background). Conversely, a given imaged scenemay include or contain a distribution of a relatively large number ofobjects of interest (targets), and a relatively small number of objectsof non-interest (corresponding to a relatively low or ‘quiet’background).

Moreover, for example, there are many hyper-spectral imaging andanalysis applications wherein the majority of hyper-spectrally imagedscenes include or contain a relatively ‘exceptionally’ small number ofobjects of interest (targets) compared to a relatively large number ofobjects of non-interest (high or noisy background). For example, suchapplications are wherein the number of objects of interest (targets),relative to the number of all objects [of interest (target) and ofnon-interest (background)] of (present or contained in) ahyper-spectrally imaged scene, corresponds to a ratio or proportion aslow as 1% [1 part per hundred (pph)], or 10⁻¹% [1 part per thousand(ppt)], or 10⁻⁴% [1 part per million (ppm)], 10⁻⁷% [1 part per billion(ppb)], or even as low as 10⁻¹⁰% [1 part per trillion (pptr)].

In addition to hyper-spectrally imaged scenes including distributions ofdifferent relative numbers (ratios, proportions) of the two maindifferent types, categories, or classes, of objects, it is noted thateach hyper-spectrally imaged object (i.e., entity, material, substance)is definable and characterizable by a set of a wide variety of numerouspossible biological, chemical, or/and physical, properties,characteristics, and behavior. For example, in a given hyper-spectrallyimaged scene, there may exist different relative numbers, and typeskinds, of objects whose ‘hyper-spectral’ image data and information(particularly including, for example, emission spectra corresponding tospectral representations in the form of spectral fingerprint orsignature pattern types of identification and characterization), arequite similar, or even nearly identical, i.e., barely distinguishable orresolvable, but whose ‘biological, chemical, or/and physical’ data andinformation (in terms of properties, characteristics, or/and behavior),are significantly different, and not at all similar or nearly identical,i.e., not at all easily distinguishable or resolvable, or vice versa.

Regardless of the actual distributions of the different relative numbers(i.e., ratios, proportions) of objects of interest (targets) and objectsof non-interest (background) in hyper-spectrally imaged scenes of asample of matter, any hyper-spectral imaging and analysis applicationultimately involves the need for identifying, distinguishing, andresolving, the objects of interest (targets) from the objects ofnon-interest (background) in the hyper-spectrally imaged scenes. Thisinvolves the need for identifying, distinguishing, and resolving, thehyper-spectral image data and information of the objects of interest(targets) from the hyper-spectral image data and information of theobjects of non-interest (background). Moreover, there is also the needfor performing such identifying, distinguishing, and resolving,procedures and operations in relation to the biological, chemical,or/and physical data and information of the objects of interest(targets) and of the objects of non-interest (background), in thehyper-spectrally imaged scenes.

Significant On-Going, Problems and Limitations of Hyper-Spectral Imagingand Analysis of a Sample of Matter

In general, significant on-going problems and limitations ofhyper-spectral imaging and analysis of a sample of matter are usuallybased on, involve, or/and are associated with, the theoretical or/andpractical difficulties and complexities that arise when performing, orattempting to perform, the previously stated three separate, butintegrated, general domains or stages, (i), (ii), and (iii), of mainactivities and procedures, with some combination of the performanceparameters of high accuracy, or/and high precision (highreproducibility), or/and high sensitivity, or/and high resolution,or/and at high speed (short time scale), be it during on-line (realtime, near-real time) or off-line, in an optimum and highly efficientmanner. Exceptional difficulties and complexities arise when performing,or attempting to perform, the general domains or stages, (i), (ii), and(iii), of main activities and procedures, with the ‘ultimate’combination of the highly desirable performance parameters of highaccuracy, ‘and’ high precision (high reproducibility), ‘and’ highsensitivity, ‘and’ high resolution, ‘and’ at high speed (short timescale), all at the same time (i.e., simultaneously), be it duringon-line (real time, near-real time) or off-line, in an optimum andhighly efficient manner.

A main source or origin of difficulties and complexities that arise whenperforming hyper-spectral imaging and analysis of a sample of matter isthe often problematic and complicating spatially or/and temporallyvarying presence of objects (entities, materials, substances) ofnon-interest (background) in the sample of matter, directly translatingto the corresponding problematic and complicating spatially or/andtemporally varying presence of objects of non-interest (background) inthe hyper-spectrally imaged scenes of the test form of the sample ofmatter. The spatially or/and temporally varying presence of objects ofnon-interest in the sample of matter negatively interferes, to a varyingextent or degree (depending upon several interdependent factors), withthe hyper-spectral imaging and analysis of the objects (entities,materials, substances) of interest (targets) in the sample of matter.Accordingly, the spatially or/and temporally varying presence of objectsof non-interest (background) in the hyper-spectrally imaged scenes ofthe test form of the sample of matter, negatively interferes, to avarying extent or degree, with the hyper-spectral imaging and analysisof objects of interest (targets) in the hyper-spectrally imaged scenesof the test form of the sample of matter.

The preceding problematic and complicating aspects, regarding thespatially or/and temporally varying presence of objects of non-interest(background), negatively affect and influence generating and collectinghyper-spectral image data and information of the sample of matter, whichin turn, negatively affect and influence processing and analyzing thegenerated and collected hyper-spectral image data and information.Moreover, such problematic and complicating aspects, along with thecorresponding negative affects and influences, subsequently make itdifficult to achieve high levels of the performance parameters ofaccuracy, precision (reproducibility), sensitivity, resolution, or/andspeed (time scale), of an overall hyper-spectral imaging and analysisapplication, such as that based on analyzing a sample of matter viahyper-spectral imaging and analysis, for identifying and characterizingan object of interest in the sample.

The preceding problematic and complicating aspects, regarding thespatially or/and temporally varying presence of objects of non-interest(background), which negatively affect and influence hyper-spectralimaging and analysis of a sample of matter, are especially relevant toan application involving on-line (real time or near-real time) oroff-line analyzing a sample of air (i.e., an air sample) viahyper-spectral imaging and analysis, for identifying and characterizingan object of interest (target) in the air sample. In particular, whereinsuch an application, the sample of air can be collected or obtained froman indoor source of air (e.g., a post office, an airport, a subwaystation, a shopping mall, a sports arena, or an office building) or froman outdoor source of air. In such an application, the interferingobjects of non-interest (background) are the numerous different(non-target) components (i.e., entities, materials, substances) presentin the air sample. In the air sample, the object of interest (target)can be a (potentially hazardous) biological agent (e.g., a bacterium[such as spore-forming bacterium Bacillus anthracis], a virus, a fungus,or a toxin), or a (potentially hazardous) chemical agent (e.g., a nerveagent [e.g., sarin, tabun, or soman], or a chemical poison [e.g., acyanide compound, or an organophosphate compound]), which is composed ormade up of organic or/and inorganic materials or substances, and ispreferably in a solid (e.g., particulate) phase.

In the collected sample of air, interfering objects of non-interest(background) originate from the numerous, spatially variable (i.e.,varying or changing with position or location) or/and temporallyvariable (i.e., varying or changing with time) different types andconcentrations of (non-target) components (i.e., entities, materials,substances) present in the source of air. The indoor or outdoor sourceof air typically includes numerous, spatially or/and temporally variabledifferent types and concentrations of (non-target) components, such asdust (fine, dry particles of matter), pollen (fine particulate orpowderlike material consisting of pollen grains produced by plants),minerals, non-target types of biological matter (mold (fungi),bacteria), and non-target types of particulate chemical matter. Such(non-target) components in the air source can be in aerosol form, beinga gaseous suspension of fine solid or liquid particles which circulatethroughout the (indoor or outdoor) air source. Such (non-target)components have relative concentrations which, typically, spatially varyor change (i.e., vary or change with position and location) or/andtemporally vary or change (i.e., vary or change with time), dependingupon the spatial or/and temporal variations in the local atmosphericenvironment and weather conditions of the indoor or outdoor source ofair, and depending upon the location and time at which the air sample iscollected or obtained from the air source. Therefore, a plurality of airsamples is expected to have such spatially or/and temporally varying(non-target) components whose relative concentrations vary in accordancewith their spatial or/and temporal variation in the source of air fromwhich the air samples are collected or obtained.

In a similar manner, an object of interest (target), such as a(potentially hazardous) biological agent (e.g., a bacterium [such asspore-forming bacterium Bacillus anthracis], a virus, a fungus, or atoxin), or a (potentially hazardous) chemical agent (e.g., a nerve agent[e.g., sarin, tabun, or soman], or a chemical poison [e.g., a cyanidecompound, or an organophosphate compound]), which is present in a sampleof air collected or obtained from an indoor or outdoor source of air,has a relative concentration which, typically, spatially varies orchanges (i.e., varies or changes with position and location) or/andtemporally varies or changes (i.e., varies or changes with time),depending upon the spatial or/and temporal variations in the localatmospheric environment and weather conditions of the indoor or outdoorsource of air, and depending upon the location and time at which the airsample is collected or obtained from the air source. Therefore, aplurality of air samples is expected to have such a spatially or/andtemporally varying object of interest (target) whose relativeconcentration varies in accordance with its spatial or/and temporalvariation in the source of air from which the air samples are collectedor obtained.

In such an application, typically, a given hyper-spectrally imaged sceneof a test form of an air sample includes or contains a distribution of arelatively small number of the object of interest (target, for example,in the form of a spectrally marked biological or chemical agent), and arelatively large number of the objects of non-interest (high or noisybackground, in the form of (non-target) components of the air sample).Moreover, in such an application, typically, the majority ofhyper-spectrally imaged scenes include or contain a relativelyexceptionally small number of the object of interest (target) comparedto a relatively large number of the objects of non-interest(background). For example, wherein the number of the object of interest(target), relative to the number of all objects [of interest (target)and of non-interest (background)] of (present or contained in) ahyper-spectrally imaged scene, corresponds to a ratio or proportion aslow as 1% [1 part per hundred (pph)], or 10⁻¹% [1 part per thousand(ppt)], or 10⁻⁴% [1 part per million (ppm)], 10⁻⁷% [1 part per billion(ppb)], or even as low as 10⁻¹⁰% [1 part per trillion (pptr)].

Additionally, in such an application, in the hyper-spectrally imagedscenes of a test form of an air sample, each hyper-spectrally imagedobject of interest (target, e.g., in the form of a spectrally markedbiological or chemical agent) and each hyper-spectrally imaged object ofnon-interest (background, in the form of (non-target) components of theair sample), is definable and characterizable by a set of a wide varietyof numerous possible biological, chemical, or/and physical, properties,characteristics, and behavior. For example, in a given hyper-spectrallyimaged scene, there may occur the scenario wherein the object ofinterest (target, in the form of a spectrally marked biological orchemical agent) and objects of non-interest (background, in the form of(non-target) components of the air sample) exhibit ‘hyper-spectral’image data and information (particularly including, for example,emission spectra corresponding to spectral representations in the formof spectral fingerprint or signature pattern types of identification andcharacterization), which are quite similar, or even nearly identical,i.e., barely distinguishable or resolvable, but whose ‘biological,chemical, or/and physical’ data and information (in terms of properties,characteristics, or/and behavior), are significantly different, and notat all similar or nearly identical, i.e., not at all easilydistinguishable or resolvable, or vice versa.

Regardless of the actual distributions of the different relative numbers(i.e., ratios, proportions) of the object of interest (target, in theform of a spectrally marked biological or chemical agent) and theobjects of non-interest (background, in the form of (non-target)components of the air sample), in the hyper-spectrally imaged scenes ofthe air sample, there ultimately is the need for identifying,distinguishing, and resolving, the object of interest (target, in theform of a spectrally marked biological or chemical agent) from theobjects of non-interest (background, in the form of (non-target)components of the air sample) in the hyper-spectrally imaged scenes.

This involves the need for identifying, distinguishing, and resolving,the hyper-spectral image data and information of the object of interest(target, in the form of a spectrally marked biological or chemicalagent) from that of the objects of non-interest (background, in the formof (non-target) components of the air sample). Moreover, there is alsothe need for performing such identifying, distinguishing, and resolving,procedures and operations in relation to the biological, chemical,or/and physical data and information, of the object of interest (target,in the form of a spectrally marked biological or chemical agent) and ofthe objects of non-interest (background, in the form of (non-target)components of the air sample) in the hyper-spectrally imaged scenes.Furthermore, there is also need for performing such identifying,distinguishing, and resolving, procedures and operations in view of thefact that the hyper-spectrally imaged scenes are generated and collectedfrom air samples wherein the objects of non-interest (background, in theform of (non-target) components of the air sample) and the object ofinterest (target, in the form of a spectrally marked biological orchemical agent) have relative concentrations that vary in accordancewith their spatial or/and temporal variation in the source of air fromwhich the air samples are collected or obtained.

Accordingly, the preceding described problematic and complicatingaspects, along with the corresponding negative affects and influences,due to the spatially or/and temporally varying presence of objects ofnon-interest (background) in a sample of air, make it difficult toachieve high levels of the performance parameters of accuracy, precision(reproducibility), sensitivity, resolution, or/and speed (time scale),of an overall hyper-spectral imaging and analysis application. This isparticularly the case for an exemplary specific application involvingon-line (real time or near-real time) or off-line hyper-spectral imagingand analysis of a sample of air (i.e., an air sample), for identifyingand characterizing an object of interest therein, wherein such anapplication, the interfering objects of non-interest (background) arethe numerous different spatially or/and temporally varying (non-target)components (i.e., entities, materials, substances) present in the airsample, and the object of interest (target) is a (potentially hazardous)biological agent (e.g., a bacterium [such as spore-forming bacteriumBacillus anthracis], a virus, a fungus, or a toxin), or a (potentiallyhazardous) chemical agent (e.g., a nerve agent [e.g., sarin, tabun, orsoman], or a chemical poison [e.g., a cyanide compound, or anorganophosphate compound]).

Thus, despite hyper-spectral imaging and analysis being well known andtaught about in the prior art and currently practiced in a wide varietyof numerous different fields and areas of science and technology, thepreceding described problematic and complicating aspects, andcorresponding negative affects and influences, which are caused by thespatially or/and temporally varying presence of objects of non-interest(background) in a sample of matter, such as a sample of air, whichsignificantly limit hyper-spectral imaging and analysis of the sample ofmatter, continue to exist, and need to be overcome.

There is thus a need for, and, it would be highly advantageous anduseful to have an invention of a method for hyper-spectral imaging andanalysis of a sample of matter, for identifying and characterizing anobject of interest therein. There is also need for such an inventionwhich also features a method for preparing a test solution or suspensionfrom a sample of matter, such that the test solution or suspension isparticularly suitable for subjecting to hyper-spectral imaging andanalysis. There is need for such an invention which is generallyapplicable for on-line (e.g., real time or near-real time) or off-linehyper-spectral imaging and analysis of various different types or kindsof samples of matter, wherein the matter, and at least one object ofinterest therein, are composed or made up of organic or/and inorganicmaterials or substances, which are in a solid (e.g., particulate) phase,a liquid (e.g., solution or suspension) phase, or/and a gaseous (e.g.,aerosol) phase. Additionally, there is need for such an invention whichprovides the capability of achieving the ‘ultimate’ combination of thehighly desirable performance parameters of high accuracy, ‘and’ highprecision (high reproducibility), ‘and’ high sensitivity, ‘and’ highresolution, ‘and’ at high speed (short time scale), all at the same time(i.e., simultaneously), be it during on-line or off-line, in an optimumand highly efficient manner.

Furthermore, there is need for such an invention which is particularlyimplementable in applications involving on-line (real time or near-realtime) or off-line hyper-spectral imaging and analysis of a sample of air(i.e., an air sample), for identifying and characterizing an object ofinterest therein, wherein the object of interest is a (potentiallyhazardous) biological agent (e.g., a bacterium [such as spore-formingbacterium Bacillus anthracis], a virus, a fungus, or a toxin), or a(potentially hazardous) chemical agent (e.g., a nerve agent [e.g.,sarin, tabun, or soman], or a chemical poison [e.g., a cyanide compound,or an organophosphate compound]). Moreover, there is need for such aninvention where the sample of air is collected or obtained (e.g., via anair sampling or collecting system) from an indoor source of air (e.g., apost office, an airport, a subway station, a shopping mall, a sportsarena, or an office building) or from an outdoor source of air.Additionally, there is particular need for such an invention wherein theobject of interest can be a biological agent, such as the spore-formingbacterium Bacillus anthracis, which is chemically marked (e.g., viaterbium trichloride [TbCl₃]), or biologically marked (e.g., viaantibodies of an immunoassay technique), for enabling identification andcharacterization thereof via hyper-spectral imaging and analysis.

SUMMARY OF THE INVENTION

The present invention relates to a method for hyper-spectral imaging andanalysis of a sample of matter, for identifying and characterizing anobject of interest therein. The present invention also features a methodfor preparing a test solution or suspension from a sample of matter,such that the test solution or suspension is particularly suitable forsubjecting to hyper-spectral imaging and analysis. The present inventionis generally applicable for on-line (e.g., real time or near-real time)or off-line hyper-spectral imaging and analysis of various differenttypes or kinds of samples of matter, wherein the matter, and at leastone object of interest therein, are composed or made up of organicor/and inorganic materials or substances, which are in a solid (e.g.,particulate) phase, a liquid (e.g., solution or suspension) phase,or/and a gaseous (e.g., aerosol) phase. The present invention providesthe capability of achieving the ‘ultimate’ combination of the highlydesirable performance parameters of high accuracy, ‘and’ high precision(high reproducibility), ‘and’ high sensitivity, ‘and’ high resolution,‘and’ at high speed (short time scale), all at the same time (i.e.,simultaneously), be it during on-line or off-line, in an optimum andhighly efficient manner.

Thus, according to an aspect of the present invention, there is provideda method for hyper-spectral imaging and analysis of a sample of matter,for identifying and characterizing an object of interest therein, themethod comprising: preparing a test solution or suspension of the sampleof matter, the preparing includes adding to the sample of matter aspectral marker specific to the object of interest, such that if theobject of interest is present in the test solution or suspension, theobject of interest when marked with the spectral marker becomes ahyper-spectrally active target which is hyper-spectrally detectable andidentifiable in the test solution or suspension; generating andcollecting hyper-spectral image data and information of the testsolution or suspension; and, processing and analyzing the hyper-spectralimage data and information, for identifying and characterizing thehyper-spectrally active target in the test solution or suspension,thereby identifying and characterizing the object of interest in thesample of matter; the method is characterized in that the step ofpreparing the test solution or suspension includes adding to the testsolution or suspension a background reducing chemical, wherein thebackground reducing chemical reduces background interfering effectscaused by presence of objects of non-interest in the test solution orsuspension, during the hyper-spectral imaging and analysis, therebyincreasing hyper-spectral detectability of the hyper-spectrally activetarget in the test solution or suspension.

According to another aspect of the present invention, there is provideda method for preparing a test solution or suspension from a sample ofmatter, the test solution or suspension being particularly suitable forsubjecting to hyper-spectral imaging and analysis, the methodcomprising: preparing a solution or suspension of the sample of matter,the preparing includes adding to the sample of matter a spectral markerspecific to an object of interest, such that if the object of interestis present in the test solution or suspension, the object of interestwhen marked with the spectral marker becomes a hyper-spectrally activetarget which is hyper-spectrally detectable and identifiable in the testsolution or suspension; characterized in that the preparing the testsolution or suspension includes adding to the solution or suspension abackground reducing chemical, thereby forming the test solution orsuspension, wherein the background reducing chemical reduces backgroundinterfering effects caused by presence of objects of non-interest in thetest solution or suspension, during hyper-spectral imaging and analysisof the test solution or suspension, thereby increasing hyper-spectraldetectability of the hyper-spectrally active target in the test solutionor suspension.

According to some embodiments of the present invention, the sample ofmatter is a sample of air.

According to some embodiments of the present invention, the object ofinterest is a biological agent or a chemical agent.

According to some embodiments of the present invention, the biologicalagent is selected from the group consisting of a bacterium, a virus, afungus, and a toxin.

According to some embodiments of the present invention, the bacterium isspore-forming bacterium Bacillus anthracis.

According to some embodiments of the present invention, the chemicalagent is selected from the group consisting of a nerve agent, and achemical poison.

According to some embodiments of the present invention, the spectralmarker specific to the object of interest is selected from the groupconsisting of chemical markers and biological markers.

According to some embodiments of the present invention, the chemicalmarker is terbium trichloride [TbCl₃].

According to some embodiments of the present invention, the biologicalmarker is an antibody of an immunoassay technique.

According to some embodiments of the present invention, the backgroundreducing chemical is selected from the group consisting of solids, andliquids.

According to some embodiments of the present invention, the liquid is anorganic liquid.

According to some embodiments of the present invention, the organicliquid is ethylene glycol (monoethylene glycol (MEG) or ethane-1,2-diol)[HOCH₂CH₂OH].

Following provision of a sample of matter, or following obtaining orcollecting a sample of matter, hyper-spectral imaging and analysis ofthe sample of matter involves the following three separate, butintegrated, general domains or stages of main activities and procedures:(i) preparing an appropriate test form (usually, a solid or liquid form)of the sample of matter, which is suitable for being subjected tohyper-spectral imaging and analysis, (ii) generating and collectinghyper-spectral image data and information of the test form of the sampleof matter, and (iii) processing and analyzing the generated andcollected hyper-spectral image data and information.

The scope of application of the present invention is primarily directedto, and focused on, the preceding stated first general domain or stageof main activities and procedures of a hyper-spectral imaging andanalysis application, i.e., being based on, and involving, preparing anappropriate test form of the sample of matter, which is suitable forbeing subjected to hyper-spectral imaging and analysis. However, intheory, and in practice, the performance parameters of accuracy,precision (reproducibility), sensitivity, resolution, or/and speed (timescale), of the first general domain or stage of main activities andprocedures of a hyper-spectral imaging and analysis application, i.e.,regarding preparation of an appropriate test form of the sample ofmatter, affect and influence the performance parameters of accuracy,precision (reproducibility), sensitivity, resolution, or/and speed (timescale), of each of the succeeding second and third general domains orstages of main activities and procedures. More specifically, mainactivities and procedures of preparing an appropriate test form of asample of matter, affect and influence generating and collectinghyper-spectral image data and information of the test form of the sampleof matter, which in turn, affect and influence processing and analyzingthe generated and collected hyper-spectral image data and information.Thus, the scope of application of the present invention also encompassesthe preceding stated second and third general domains or stages of mainactivities and procedures of hyper-spectral imaging and analysis of asample of matter.

A main aspect of novelty and inventiveness of the present invention isthat in the method for hyper-spectral imaging and analysis of a sampleof matter, for identifying and characterizing an object of interesttherein, the main step (procedure) of preparing the test solution orsuspension of the sample of matter includes the unique and criticallyimportant step (procedure) of adding to the test solution or suspensiona background reducing chemical. The background reducing chemical reducesbackground interfering effects caused by presence of objects ofnon-interest in the test solution or suspension, during thehyper-spectral imaging and analysis, thereby increasing hyper-spectraldetectability of the hyper-spectrally active target in the test solutionor suspension.

Another main aspect of novelty and inventiveness of the presentinvention is that in the method for preparing a test solution orsuspension from a sample of matter, the test solution or suspensionbeing particularly suitable for subjecting to hyper-spectral imaging andanalysis, the main step (procedure) of preparing a solution orsuspension of the sample of matter includes the unique and criticallyimportant step (procedure) of adding to the solution or suspension abackground reducing chemical. This results in forming the test solutionor suspension, wherein the background reducing chemical reducesbackground interfering effects caused by presence of objects ofnon-interest in the test solution or suspension, during hyper-spectralimaging and analysis of the test solution or suspension, therebyincreasing hyper-spectral detectability of the hyper-spectrally activetarget in the test solution or suspension.

An exemplary specific application of the present invention involveson-line (real time or near-real time) or off-line hyper-spectral imagingand analysis of a sample of air (i.e., an air sample), for identifyingand characterizing an object of interest therein, wherein the object ofinterest is a (potentially hazardous) biological agent or a (potentiallyhazardous) chemical agent. In general, the object of interest (i.e.,biological agent or chemical agent) in the air sample is composed ormade up of organic or/and inorganic materials or substances, which arein a solid (e.g., particulate) phase, a liquid (e.g., solution orsuspension) phase, or/and a gaseous (e.g., aerosol) phase. Preferably,the object of interest (i.e., biological agent or chemical agent) in theair sample is composed or made up of organic or/and inorganic materialsor substances, which are in a particulate form solid phase or/and arepresent (e.g., absorbed or/and adsorbed) on particles of the air sample.The sample of air is collected or obtained (e.g., via a standard type ofair sampling or collecting system) from an indoor source of air (e.g., apost office, an airport, a subway station, a shopping mall, a sportsarena, or an office building), or from an outdoor source of air.Exemplary biological agents are bacteria, viruses, fungi, and toxins.Exemplary chemical agents are nerve agents (e.g., sarin, tabun, andsoman), and chemical poisons (e.g., cyanide compounds, andorganophosphate compounds). The object of interest can be a biologicalagent, such as the (extremely hazardous) spore-forming bacteriumBacillus anthracis, which is chemically marked (e.g., via terbiumtrichloride [TbCl₃]), or biologically marked (e.g., via antibodies of animmunoassay technique), as part of the main step (procedure) ofpreparing a test solution or suspension of the sample of matter, i.e.,the air sample, for enabling identification and characterization of theobject of interest via hyper-spectral imaging and analysis.

In such an exemplary specific application of the present invention, itwas empirically determined (see the Examples hereinbelow) that thebackground reducing chemical is, preferably, an organic liquid, such asethylene glycol (equivalently known as monoethylene glycol (MEG) orethane-1,2-diol) [HOCH₂CH₂OH]. The specific type or kind of backgroundreducing chemical, i.e., the ethylene glycol (MEG) [HOCH₂CH₂OH], isselected such that the background reducing chemical effectively (i.e.,measurably) reduces (decreases) background interfering effects caused bythe presence of the numerous, different types of objects of non-interest(background), i.e., (non-target) components, present in the testsolution or suspension (particularly the numerous, spatially or/andtemporally variable different types and concentrations of (non-target)components, such as dust, pollen, minerals, non-target types ofbiological matter (mold (fungi), bacteria), and non-target types ofparticulate chemical matter, which originated from the air sample),during the hyper-spectral imaging and analysis of the test solution orsuspension. This results in increasing (enhancing) hyper-spectraldetectability of the hyper-spectrally active target (i.e., the{biological agent Bacillus anthracis spore (dipicolinic acid[DPA])—terbium trichloride [TbCl₃]} complex, or the {biological agentBacillus anthracis spore antigen—antibody} complex) in the test solutionor suspension, during the hyper-spectral imaging and analysis of thetest solution or suspension. Accordingly, addition of the backgroundreducing chemical, i.e., the ethylene glycol (MEG) [HOCH₂CH₂OH], to thetest solution or suspension of the air sample results in increasing(enhancing) hyper-spectral detectability of the (target) spore-formingbacterium Bacillus anthracis, present in the air sample.

The present invention is implemented by performing steps or procedures,and sub-steps or sub-procedures, in a manner selected from the groupconsisting of manually, semi-automatically, fully automatically, and acombination thereof, involving use and operation of system units, systemsub-units, devices, assemblies, sub-assemblies, mechanisms, structures,components, and elements, and, peripheral equipment, utilities,accessories, chemical reagents, and materials, in a manner selected fromthe group consisting of manually, semi-automatically, fullyautomatically, and a combination thereof. Moreover, according to actualsteps or procedures, sub-steps or sub-procedures, system units, systemsub-units, devices, assemblies, sub-assemblies, mechanisms, structures,components, and elements, and, peripheral equipment, utilities,accessories, chemical reagents, and materials, used for implementing aparticular embodiment of the disclosed invention, the steps orprocedures, and sub-steps or sub-procedures, are performed by usinghardware, software, or/and an integrated combination thereof, and thesystem units, sub-units, devices, assemblies, sub-assemblies,mechanisms, structures, components, and elements, and, peripheralequipment, utilities, accessories, and materials, operate by usinghardware, software, or/and an integrated combination thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the present invention are herein described, by way ofexample only, with reference to the accompanying drawings. With specificreference now to the drawings in detail, it is stressed that theparticulars shown are by way of example and for purposes of illustrativedescription of embodiments of the present invention. In this regard, thedescription taken together with the accompanying drawings make apparentto those skilled in the art how the embodiments of the present inventionmay be practiced.

In the drawings:

FIG. 1 is a flow diagram of a preferred embodiment of the main steps orprocedures of the method for hyper-spectral imaging and analysis of asample of matter, for identifying and characterizing an object ofinterest therein, in accordance with the present invention;

FIGS. 2 a and 2 b are exemplary empirically determined graphical plots(spectra) of Emission Intensity (normalized) as a function of EmissionWavelength (nm) of exemplary dust particles 1 and 2, respectively, indry form, subjected to hyper-spectral imaging and analysis, showing thespectral fingerprints (SFPs) thereof, as described hereinbelow inExample 1, in accordance with the present invention;

FIG. 3 a is an exemplary empirically determined graphical plot(spectrum) of Emission Intensity (normalized) as a function of EmissionWavelength (mm) of a background-only test suspension [containing onlyobjects of non-interest suspended in the exemplary background reducingchemical being the organic liquid ethylene glycol (monoethylene glycol(MEG)); absent of any object of interest or target (i.e., absent of ahyper-spectrally active target being the exemplary {biological agentBacillus subtilis spore (dipicolinic acid [DPA])—terbium trichloride[TbCl₃]} complex], subjected to hyper-spectral imaging and analysis,showing the spectral fingerprint (SFP) thereof, as described hereinbelowin Example 3, in accordance with the present invention;

FIG. 3 b is an exemplary empirically determined graphical plot(spectrum) of Emission Intensity (normalized) as a function of EmissionWavelength (nm) of a target-containing test suspension [includingobjects of non-interest, and an exemplary object of interest or target(i.e., a hyper-spectrally active target being the exemplary {biologicalagent Bacillus subtilis spore (dipicolinic acid [DPA])—terbiumtrichloride [TbCl₃]} complex, suspended in the exemplary backgroundreducing chemical being the organic liquid ethylene glycol (monoethyleneglycol (MEG))], subjected to hyper-spectral imaging and analysis,showing the spectral fingerprints (SFPs) thereof, as describedhereinbelow in Example 3, in accordance with the present invention;

FIG. 4 a is an exemplary empirically determined bar graph of PositivePixels (%) as a function of Spore Count (absolute number) showingdistribution of ‘background’ and ‘target’ spectral fingerprints (SFPs)among Positive Pixels [of the emission peak (540±5 nm) of an exemplaryobject of interest or target (i.e., a hyper-spectrally active targetbeing the exemplary {biological agent Bacillus subtilis spore(dipicolinic acid [DPA])—terbium trichloride [TbCl₃]} complex], as afunction of Spore Count, based on hyper-spectral image data andinformation obtained from FIGS. 3 a and 3 b and similar experiments, asdescribed hereinbelow in Example 3, in accordance with the presentinvention; and

FIG. 4 b is an exemplary empirically determined bar graph of PositivePixels (%) as a function of Spore Count (absolute number) showing‘reproducibility’ of the distribution of ‘background’ and ‘target’spectral fingerprints (SFPs) among Positive Pixels [of the emission peak(540±5 nm) of an exemplary object of interest or target (i.e., ahyper-spectrally active target being the exemplary {biological agentBacillus subtilis spore (dipicolinic acid [DPA])—terbium trichloride[TbCl₃]} complex], as a function of Spore Count, based on hyper-spectralimage data and information obtained by repeating experiments of Example3, as described hereinbelow in Example 3, in accordance with the presentinvention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention relates to a method for hyper-spectral imaging andanalysis of a sample of matter, for identifying and characterizing anobject of interest therein. The present invention also features a methodfor preparing a test solution or suspension from a sample of matter,such that the test solution or suspension is particularly suitable forsubjecting to hyper-spectral imaging and analysis. The present inventionis generally applicable for on-line (e.g., real time or near-real time)or off-line hyper-spectral imaging and analysis of various differenttypes or kinds of samples of matter, wherein the matter, and at leastone object of interest therein, are composed or made up of organicor/and inorganic materials or substances, which are in a solid (e.g.,particulate) phase, a liquid (e.g., solution or suspension) phase,or/and a gaseous (e.g., aerosol) phase. The present invention providesthe capability of achieving the ‘ultimate’ combination of the highlydesirable performance parameters of high accuracy, ‘and’ high precision(high reproducibility), ‘and’ high sensitivity, ‘and’ high resolution,‘and’ at high speed (short time scale), all at the same time (i.e.,simultaneously), be it during on-line or off-line, in an optimum andhighly efficient manner.

A first main aspect of the present invention is provision of a methodfor hyper-spectral imaging and analysis of a sample of matter, foridentifying and characterizing an object of interest therein, the methodincluding the following main steps or procedures, and, components andfunctionalities thereof: preparing a test solution or suspension of thesample of matter, the preparing includes adding to the sample of mattera spectral marker specific to the object of interest, such that if theobject of interest is present in the test solution or suspension, theobject of interest when marked with the spectral marker becomes ahyper-spectrally active target which is hyper-spectrally detectable andidentifiable in the test solution or suspension; generating andcollecting hyper-spectral image data and information of the testsolution or suspension; and, processing and analyzing the hyper-spectralimage data and information, for identifying and characterizing thehyper-spectrally active target in the test solution or suspension,thereby identifying and characterizing the object of interest in thesample of matter; the method is characterized in that the step ofpreparing the test solution or suspension includes adding to the testsolution or suspension a background reducing chemical, wherein thebackground reducing chemical reduces background interfering effectscaused by presence of objects of non-interest in the test solution orsuspension, during the hyper-spectral imaging and analysis, therebyincreasing hyper-spectral detectability of the hyper-spectrally activetarget in the test solution or suspension.

A second main aspect of the present invention is provision of a methodfor preparing a test solution or suspension from a sample of matter, thetest solution or suspension being particularly suitable for subjectingto hyper-spectral imaging and analysis, the method including thefollowing main steps or procedures, and, components and functionalitiesthereof: preparing a solution or suspension of the sample of matter, thepreparing includes adding to the sample of matter a spectral markerspecific to an object of interest, such that if the object of interestis present in the test solution or suspension, the object of interestwhen marked with the spectral marker becomes a hyper-spectrally activetarget which is hyper-spectrally detectable and identifiable in the testsolution or suspension; characterized in that preparing the solution orsuspension includes adding to the solution or suspension a backgroundreducing chemical, thereby forming the test solution or suspension,wherein the background reducing chemical reduces background interferingeffects caused by presence of objects of non-interest in the testsolution or suspension, during hyper-spectral imaging and analysis ofthe test solution or suspension, thereby increasing hyper-spectraldetectability of the hyper-spectrally active target in the test solutionor suspension.

Following provision of a sample of matter, or following obtaining orcollecting a sample of matter, hyper-spectral imaging and analysis ofthe sample of matter involves the following three separate, butintegrated, general domains or stages of main activities and procedures:(i) preparing an appropriate test form (usually, a solid or liquid form)of the sample of matter, which is suitable for being subjected tohyper-spectral imaging and analysis, (ii) generating and collectinghyper-spectral image data and information of the test form of the sampleof matter, and (iii) processing and analyzing the generated andcollected hyper-spectral image data and information.

The scope of application of the present invention is primarily directedto, and focused on, the preceding stated first general domain or stageof main activities and procedures of a hyper-spectral imaging andanalysis application, i.e., being based on, and involving, preparing anappropriate test form of the sample of matter, which is suitable forbeing subjected to hyper-spectral imaging and analysis. However, intheory, and in practice, the performance parameters of accuracy,precision (reproducibility), sensitivity, resolution, or/and speed (timescale), of the first general domain or stage of main activities andprocedures of a hyper-spectral imaging and analysis application, i.e.,regarding preparation of an appropriate test form of the sample ofmatter, affect and influence the performance parameters of accuracy,precision (reproducibility), sensitivity, resolution, or/and speed (timescale), of each of the succeeding second and third general domains orstages of main activities and procedures. More specifically, mainactivities and procedures of preparing an appropriate test form of asample of matter, affect and influence generating and collectinghyper-spectral image data and information of the test form of the sampleof matter, which in turn, affect and influence processing and analyzingthe generated and collected hyper-spectral image data and information.Thus, the scope of application of the present invention also encompassesthe preceding stated second and third general domains or stages of mainactivities and procedures of hyper-spectral imaging and analysis of asample of matter.

Embodiments of the present invention include several special technicalfeatures, and, aspects of novelty and inventiveness over prior artteachings in the relevant fields and arts of the invention.

A special technical feature of embodiments of the present invention isthat in the method for hyper-spectral imaging and analysis of a sampleof matter, for identifying and characterizing an object of interesttherein, the main step (procedure) of preparing the test solution orsuspension of the sample of matter includes the unique and criticallyimportant step (procedure) of adding to the test solution or suspensiona background reducing chemical. The background reducing chemical reducesbackground interfering effects caused by presence of objects ofnon-interest in the test solution or suspension, during thehyper-spectral imaging and analysis, thereby increasing hyper-spectraldetectability of the hyper-spectrally active target in the test solutionor suspension.

Another special technical feature of embodiments of the presentinvention is that in the method for preparing a test solution orsuspension from a sample of matter, the test solution or suspensionbeing particularly suitable for subjecting to hyper-spectral imaging andanalysis, the main step (procedure) of preparing a solution orsuspension of the sample of matter includes the unique and criticallyimportant step (procedure) of adding to the solution or suspension abackground reducing chemical. This results in forming the test solutionor suspension, wherein the background reducing chemical reducesbackground interfering effects caused by presence of objects ofnon-interest in the test solution or suspension, during hyper-spectralimaging and analysis of the test solution or suspension, therebyincreasing hyper-spectral detectability of the hyper-spectrally activetarget in the test solution or suspension.

An exemplary specific application of the present invention involveson-line (real time or near-real time) or off-line hyper-spectral imagingand analysis of a sample of air (i.e., an air sample), for identifyingand characterizing an object of interest therein, wherein the object ofinterest is a (potentially hazardous) biological agent or a (potentiallyhazardous) chemical agent. In general, the object of interest (i.e.,biological agent or chemical agent) in the air sample is composed ormade up of organic or/and inorganic materials or substances, which arein a solid (e.g., particulate) phase, a liquid (e.g., solution orsuspension) phase, or/and a gaseous (e.g., aerosol) phase. Preferably,the object of interest (i.e., biological agent or chemical agent) in theair sample is composed or made up of organic or/and inorganic materialsor substances, which are in a particulate form solid phase or/and arepresent (e.g., absorbed or/and adsorbed) on particles of the air sample.The sample of air is collected or obtained (e.g., via a standard type ofair sampling or collecting system) from an indoor source of air (e.g., apost office, an airport, a subway station, a shopping mall, a sportsarena, or an office building), or from an outdoor source of air.Exemplary biological agents are bacteria, viruses, fingi, and toxins.Exemplary chemical agents are nerve agents (e.g., sarin, tabun, andsoman), and chemical poisons (e.g., cyanide compounds, andorganophosphates). The object of interest can be a biological agent,such as the (extremely hazardous) spore-forming bacterium Bacillusanthracis, which is chemically marked (e.g., via terbium trichloride[TbCl₃]), or biologically marked (e.g., via antibodies of an immunoassaytechnique), for enabling identification and characterization of theobject of interest via hyper-spectral imaging and analysis.

In such an exemplary specific application of the present invention, itwas empirically determined (see the Examples hereinbelow) that thebackground reducing chemical is, preferably, an organic liquid, such asethylene glycol (equivalently known as monoethylene glycol (MEG) orethane-1,2-diol) [HOCH₂CH₂OH]. The specific type or kind of backgroundreducing chemical, i.e., the ethylene glycol (MEG) [HOCH₂CH₂OH], isselected such that the background reducing chemical effectively (i.e.,measurably) reduces (decreases) background interfering effects caused bythe presence of the numerous, different types of objects of non-interest(background), i.e., (non-target) components, present in the testsolution or suspension (particularly the numerous, spatially or/andtemporally variable different types and concentrations of (non-target)components, such as dust, pollen, minerals, non-target types ofbiological matter (mold (fungi), bacteria), and non-target types ofparticulate chemical matter, which originated from the sample ofmatter), during the hyper-spectral imaging and analysis of the testsolution or suspension. This results in increasing (enhancing)hyper-spectral detectability of the hyper-spectrally active target(i.e., the {biological agent Bacillus anthracis spore (dipicolinic acid[DPA])—terbium trichloride [TbCl₃]} complex], or the {biological agentBacillus anthracis spore antigen—antibody} complex) in the test solutionor suspension, during the hyper-spectral imaging and analysis of thetest solution or suspension. Accordingly, addition of the backgroundreducing chemical, i.e., the ethylene glycol (MEG) [HOCH₂CH₂OH], to thetest solution or suspension of the air sample results in increasing(enhancing) hyper-spectral detectability of the (target) spore-formingbacterium Bacillus anthracis, present in the air sample.

It is to be understood that the present invention is not limited in itsapplication to the details of the order or sequence, and number, ofsteps or procedures, sub-steps or sub-procedures, of operation orimplementation of the method, or to the details of the equipment,chemical reagents, and materials, used for implementing the method, setforth to in the following illustrative description, accompanyingdrawings, and examples, unless otherwise specifically stated herein.Moreover, although illustrative description, and examples, of thepresent invention are primarily focused on applications involving abiological agent, wherein the biological agent is, for example, the(extremely hazardous) spore-forming bacterium Bacillus anthracis, whichis chemically marked (e.g., via terbium trichloride [TbCl₃]), orbiologically marked (e.g., via antibodies of an immunoassay technique),for enabling identification and characterization thereof viahyper-spectral imaging and analysis, it is to be fully understood thatthe present invention is also applicable to other biological agents,such as other bacteria, viruses, fungi, and toxins, and the presentinvention is also applicable to chemical agents, such as nerve agents(e.g., sarin, tabun, and soman), and chemical poisons (e.g., cyanidecompounds, and organophosphates). Accordingly, the present invention canbe practiced or implemented according to various other alternativeembodiments and in various other alternative ways.

It is also to be understood that all technical and scientific words,terms, or/and phrases, used herein throughout the present disclosurehave either the identical or similar meaning as commonly understood byone of ordinary skill in the art to which this invention belongs, unlessotherwise specifically defined or stated herein. Phraseology,terminology, and, notation, employed herein throughout the presentdisclosure are for the purpose of description and should not be regardedas limiting.

For example, in the illustrative description of the present invention,there is general reference to the terms ‘object’ and ‘objects’, in orderto illustrate implementation of the present invention. Herein, the term‘object’ as used for illustratively describing the present invention isconsidered equivalent to, and synonymous with, at least part of anentity, material, substance, or structure, which, singly or incombination with other objects (entities, materials, substances, orstructures), typically as part of a scene (defined hereinbelow), issubjected to a hyper-spectral imaging process or technique. In general,such an object is definable and characterizable by a set of a widevariety of numerous possible biological, chemical, or/and physical,properties, characteristics, and behavior. Also, for example, in theillustrative description of the present invention, there is generalreference to the term ‘marked’ in the phrase ‘spectrally marked’, and,to the term ‘marker’ in the phrases ‘spectral marker’, ‘chemicalmarker’, and ‘biological marker’, in order to illustrate implementationof the present invention. As used herein, the terms ‘marked’ and‘marker’ are considered equivalent to, and synonymous with, the terms‘labeled’ and ‘label’, respectively. Thus, herein, the phrases‘spectrally marked’, ‘spectral marker’, ‘chemical marker’, and‘biological marker’, are considered equivalent to, and synonymous with,the phrases ‘spectrally labeled’, ‘spectral label’, ‘chemical label’,and ‘biological label’, respectively.

Moreover, all technical and scientific words, terms, or/and phrases,introduced, defined, described, or/and exemplified, in the above Fieldand Background section, are equally or similarly applicable in theillustrative description of the preferred embodiments, examples, andappended claims, of the present invention. Immediately following areselected definitions and exemplary usages of words, terms, or/andphrases, which are used throughout the illustrative description of thepreferred embodiments, examples, and appended claims, of the presentinvention, and are especially relevant for understanding thereof.

Each of the following terms: ‘includes’, ‘including’, ‘has’, ‘having’,‘comprises’, and ‘comprising’, and, their derivatives and conjugates,means ‘including, but not limited to’.

Each of the following terms written in singular grammatical form: ‘a’,‘an’, and ‘the’, may also refer to, and encompass, a plurality of thestated entity or object, unless otherwise specifically defined or statedherein, or, unless the context clearly dictates otherwise. For example,the phrases ‘an object’, ‘a target’, ‘a component’, and ‘an element’,may also refer to, and encompass, a plurality of objects, a plurality oftargets, a plurality of components, and a plurality of elements,respectively.

The term ‘about’ refers to ±10% of the stated numerical value

The phrase ‘room temperature’ refers to a temperature in a range ofbetween about 20° C. and about 25° C.

Throughout the illustrative description of the embodiments, theexamples, and the appended claims, of the present invention, a numericalvalue of a parameter, feature, object, or dimension, may be stated ordescribed in terms of a numerical range format. It is to be fullyunderstood that the stated numerical range format is provided forillustrating implementation of the present invention, and is not to beunderstood or construed as inflexibly limiting the scope of the presentinvention.

Accordingly, a stated or described numerical range also refers to, andencompasses, all possible sub-ranges and individual numerical values(where a numerical value may be expressed as a whole, integral, orfractional number) within that stated or described numerical range. Forexample, a stated or described numerical range ‘from 1 to 6’ also refersto, and encompasses, all possible sub-ranges, such as ‘from 1 to 3’,‘from 1 to 4’, ‘from 1 to 5’, ‘from 2 to 4’, ‘from 2 to 6’, ‘from 3 to6’, etc., and individual numerical values, such as ‘1’, ‘1.3’, ‘2’,‘2.8’, ‘3’, ‘3.5’, ‘4’, ‘4.6’, ‘5’, ‘5.2’, and ‘6’, within the stated ordescribed numerical range of ‘from 1 to 6’. This applies regardless ofthe numerical breadth, extent, or size, of the stated or describednumerical range.

Moreover, for stating or describing a numerical range, the phrase ‘in arange of between about a first numerical value and about a secondnumerical value’, is considered equivalent to, and meaning the same as,the phrase ‘in a range of from about a first numerical value to about asecond numerical value’, and, thus, the two equivalently meaning phrasesmay be used interchangeably. For example, for stating or describing thenumerical range of room temperature, the phrase ‘room temperature refersto a temperature in a range of between about 20° C. and about 25° C.’,is considered equivalent to, and meaning the same as, the phrase ‘roomtemperature refers to a temperature in a range of from about 20° C. toabout 25° C.’.

Steps or procedures, sub-steps or sub-procedures, and, equipment andmaterials, system units, system sub-units, devices, assemblies,sub-assemblies, mechanisms, structures, components, elements, andconfigurations, and, peripheral equipment, utilities, accessories,chemical reagents, and materials, as well as operation andimplementation, of exemplary preferred embodiments, alternativepreferred embodiments, specific configurations, and, additional andoptional aspects, characteristics, or features, thereof, according tothe present invention, are better understood with reference to thefollowing illustrative description and accompanying drawings.

According to the first main aspect of the present invention, there isprovision of a method for hyper-spectral imaging and analysis of asample of matter, for identifying and characterizing an object ofinterest therein.

Referring now to the drawings, FIG. 1 is a flow diagram of a preferredembodiment of the main steps or procedures, and, components andfunctionalities thereof, of the method for hyper-spectral imaging andanalysis of a sample of matter, for identifying and characterizing anobject of interest therein. Accordingly, the method includes thefollowing main steps or procedures, and, components and functionalitiesthereof: preparing a test solution or suspension of the sample ofmatter, the preparing includes adding to the sample of matter a spectralmarker specific to the object of interest, such that if the object ofinterest is present in the test solution or suspension, the object ofinterest when marked with the spectral marker becomes a hyper-spectrallyactive target which is hyper-spectrally detectable and identifiable inthe test solution or suspension; generating and collectinghyper-spectral image data and information of the test solution orsuspension; and, processing and analyzing the hyper-spectral image dataand information, for identifying and characterizing the hyper-spectrallyactive target in the test solution or suspension, thereby identifyingand characterizing the object of interest in the sample of matter.

The method is characterized in that the step of preparing the testsolution or suspension includes adding to the test solution orsuspension a background reducing chemical, wherein the backgroundreducing chemical reduces background interfering effects caused bypresence of objects of non-interest in the test solution or suspension,during the hyper-spectral imaging and analysis, thereby increasinghyper-spectral detectability of the hyper-spectrally active target inthe test solution or suspension.

Applicable Types or Kinds, and Forms, of the Sample of Matter

In general, the present invention is applicable to essentially any typeor kind, and form, of sample of matter. The sample of matter isgenerally a relatively small quantity of matter which is representativeof, and an example of, (i.e., a sample), of a relatively large, quantityof the matter, where the matter is generally something (i.e., entity,material, substance) that has mass, occupies volume, and exists as asolid, liquid, gas, or a combination thereof. The sample of matter mayalso be considered as being a specimen (i.e., example) of the matter.The sample of matter is composed or made up of any number, and type orkind, of objects, wherein each object generally refers to, and isconsidered equivalent to, and synonymous with, at least part of thematter, and therefore, that which is present in a sample of the matter.Accordingly, each object generally refers to, and is consideredequivalent to, and synonymous with, at least part of something (i.e.,entity, material, substance) that has mass, occupies volume, and existsas a solid, liquid, gas, or a combination thereof. Moreover, each object(i.e., at least part of the matter) is definable and characterizable bya set of a wide variety of numerous possible biological, chemical,or/and physical, properties, characteristics, and behavior.

>For a Sample of Air (Air Sample) Containing Numerous DifferentSpatially or/and Temporally Varying Interfering Objects of Non-Interest(Background), and a Biological Agent or Chemical Agent Type of Object ofInterest (Target)<

In an exemplary specific embodiment of the present invention, the sampleof matter is a sample of air (i.e., an air sample). Accordingly, anexemplary specific embodiment of the present invention involves on-line(real time or near-real time) or off-line hyper-spectral imaging andanalysis of a sample of air (i.e., an air sample), for identifying andcharacterizing an object of interest therein.

An exemplary specific application of the present invention involveson-line (real time or near-real time) or off-line hyper-spectral imagingand analysis of a sample of air (i.e., an air sample), for identifyingand characterizing an object of interest therein, wherein the object ofinterest is a biological agent or a chemical agent. In general, theobject of interest (i.e., biological agent or chemical agent) in the airsample is composed or made up of organic or/and inorganic materials orsubstances, which are in a solid (e.g., particulate) phase, a liquid(e.g., solution or suspension) phase, or/and a gaseous (e.g., aerosol)phase. Preferably, the object of interest (i.e., biological agent orchemical agent) in the air sample is composed or made up of organicor/and inorganic materials or substances, which are in a particulateform solid phase or/and are present (e.g., absorbed or/and adsorbed) onparticles of the air sample. The sample of air is collected or obtained(e.g., via a standard type of air sampling or collecting system) from anindoor source of air (e.g., a post office, an airport, a subway station,a shopping mall, a sports arena, or an office building), or from anoutdoor source of air. Exemplary biological agents are bacteria,viruses, fungi, and toxins. Exemplary chemical agents are nerve agents(e.g., sarin, tabun, and soman), and chemical poisons (e.g., cyanidecompounds, and organophosphate compounds). The object of interest can bea biological agent, such as the spore-forming bacterium Bacillusanthracis, which is chemically marked (e.g., via terbium trichloride[TbCl₃]), or biologically marked (e.g., via antibodies of an immunoassaytechnique), as part of the main step (procedure) of preparing a testsolution or suspension of the sample of matter, i.e., the air sample,for enabling identification and characterization of the object ofinterest via hyper-spectral imaging and analysis.

Types, Categories, or Classes, of Objects in the Sample of Matter

In general, in the sample of matter (including the objects andcomponents thereof present in the sample of matter), the objects (i.e.,entities, materials, substances) are typed, categorized, or classified,according to two main different types, categories, or classes. Namely,‘objects of non-interest’, and ‘objects of interest’, each of which isbasically defined as follows. ‘Objects of non-interest’ correspond toobjects of (present or contained in) the sample of matter which are notof interest to a human operator (observer, viewer, analyzer, or/andcontroller) of a process involving the sample of matter. ‘Objects ofinterest’ correspond to objects of (present or contained in) the sampleof matter which are of interest to a human operator of a processinvolving the sample of matter. For further understanding thesignificantly different meanings and attributes of objects ofnon-interest and objects of interest, in the context of the presentinvention, objects of non-interest are considered as being part of the‘background’ of, or within, the sample of matter, whereas objects ofinterest are considered as being ‘targets’ of, or within, the sample ofmatter. Accordingly, individual objects among a plurality, collection,or ensemble, of several objects (i.e., entities, materials, substances)of (present or contained in) the sample of matter, are typed,categorized, or classified, according to the above stated two maindifferent types, categories, or classes, of objects, i.e., objects ofnon-interest (i.e., background), and objects of interest (i.e.,targets).

Typically, the sample of matter includes or contains a distribution ofdifferent relative numbers (i.e., ratios, proportions) of the precedingdefined two main different types, categories, or classes, of objects.For example, the sample of matter may include or contain a distributionof a relatively small number of objects of interest (targets), and arelatively large number of objects of non-interest (corresponding to arelatively high or ‘noisy’ background). Conversely, the sample of mattermay include or contain a distribution of a relatively large number ofobjects of interest (targets), and a relatively small number of objectsof non-interest (corresponding to a relatively low or ‘quiet’background).

Moreover, for example, there are many applications of the presentinvention wherein the sample of matter includes or contains a relatively‘exceptionally’ small number of objects of interest (targets) comparedto a relatively large number of objects of non-interest (high or noisybackground). For example, such applications are wherein the number ofobjects of interest (targets), relative to the number of all objects [ofinterest (target) and of non-interest (background)] of (present orcontained in) the sample of matter, corresponds to a ratio or proportionas low as 1% [1 part per hundred (pph)], or 10⁻¹% [1 part per thousand(ppt)], or 1% [1 part per million (ppm)], 10⁻⁷% [1 part per billion(ppb)], or even as low as 10⁻¹⁰% [1 part per trillion (pptr)].

In addition to the sample of matter including distributions of differentrelative numbers (ratios, proportions) of the two main different types,categories, or classes, of objects, it is noted that, as indicatedhereinabove, each object (i.e., entity, material, substance) isdefinable and characterizable by a set of a wide variety of numerouspossible biological, chemical, or/and physical, properties,characteristics, and behavior. For example, in the sample of matter,there may exist different types, kinds, and numbers, of objects whose‘hyper-spectral’ image data and information (including, for example,emission spectra corresponding to spectral representations in the formof spectral fingerprint (herein, abbreviated, and also referred to, asSFP) or signature pattern types of identification and characterization),are quite similar, or even nearly identical, i.e., barelydistinguishable or resolvable, but whose ‘biological, chemical, or/andphysical’ data and information (in terms of properties, characteristics,or/and behavior), are significantly different, and not at all similar ornearly identical, i.e., not at all easily distinguishable or resolvable,or vice versa.

Regardless of the actual distributions of the different relative numbers(i.e., ratios, proportions) of objects of interest (targets) and objectsof non-interest (background) in the sample of matter, any application ofthe present invention ultimately involves the need for identifying,distinguishing, and resolving, the objects of interest (targets) fromthe objects of non-interest (background) in the sample of matter. Thisinvolves the need for identifying, distinguishing, and resolving, thehyper-spectral image data and information of the objects of interest(targets) from the hyper-spectral image data and information of theobjects of non-interest (background). Moreover, there is also the needfor performing such identifying, distinguishing, and resolving,procedures and operations in relation to the biological, chemical,or/and physical data and information of the objects of interest(targets) and of the objects of non-interest (background), in the sampleof matter.

A main source or origin of difficulties and complexities that arise whenperforming hyper-spectral imaging and analysis of a sample of matter isthe often problematic and complicating spatially or/and temporallyvarying presence of objects (entities, materials, substances) ofnon-interest (background) in the sample of matter, directly translatingto the corresponding problematic and complicating spatially or/andtemporally varying presence of objects of non-interest (background) inthe hyper-spectrally imaged scenes of the test form of the sample ofmatter. The spatially or/and temporally varying presence of objects ofnon-interest in the sample of matter negatively interferes, to a varyingextent or degree (depending upon several interdependent factors), withthe hyper-spectral imaging and analysis of the objects (entities,materials, substances) of interest (targets) in the sample of matter.Accordingly, the spatially or/and temporally varying presence of objectsof non-interest (background) in the hyper-spectrally imaged scenes ofthe test form of the sample of matter, negatively interferes, to avarying extent or degree, with the hyper-spectral imaging and analysisof objects of interest (targets) in the hyper-spectrally imaged scenesof the test form of the sample of matter.

The preceding problematic and complicating aspects, regarding thespatially or/and temporally varying presence of objects of non-interest(background), negatively affect and influence generating and collectinghyper-spectral image data and information of the sample of matter, whichin turn, negatively affect and influence processing and analyzing thegenerated and collected hyper-spectral image data and information.Moreover, such problematic and complicating aspects, along with thecorresponding negative affects and influences, subsequently make itdifficult to achieve high levels of the performance parameters ofaccuracy, precision (reproducibility), sensitivity, resolution, or/andspeed (time scale), of an overall hyper-spectral imaging and analysisapplication, such as that based on analyzing a sample of matter viahyper-spectral imaging and analysis, for identifying and characterizingan object of interest in the sample.

>For a Sample of Air (Air Sample) Containing Numerous DifferentSpatially or/and Temporally Varying Interfering Objects of Non-Interest(Background), and a Biological Agent or Chemical Agent Type of Object ofInterest (Target)<

The preceding problematic and complicating aspects, regarding thespatially or/and temporally varying presence of objects of non-interest(background), which negatively affect and influence hyper-spectralimaging and analysis of a sample of matter, are especially relevant toan application of the present invention, involving on-line (real time ornear-real time) or off-line analyzing a sample of air (i.e., an airsample) via hyper-spectral imaging and analysis, for identifying andcharacterizing an object of interest (target) in the air sample. Inparticular, wherein such an application, the sample of air is collectedor obtained (e.g., via a standard type of air sampling or collectingsystem) from an indoor source of air (e.g., a post office, an airport, asubway station, a shopping mall, a sports arena, or an office building)or from an outdoor source of air.

In such an application, the interfering objects of non-interest(background) are the numerous different (non-target) components (i.e.,entities, materials, substances) present in the air sample. In the airsample, the object of interest (target) is a (potentially hazardous)biological agent (e.g., bacterium [such as (the extremely hazardous)spore-forming bacterium Bacillus anthracis], a virus, a fungus, or atoxin), or a (potentially hazardous) chemical agent (e.g., a nerve agent[e.g., sarin, tabun, or soman], or a chemical poison [e.g., a cyanidecompound, or an organophosphate compound]), which is composed or made upof organic or/and inorganic materials or substances, and is preferablyin a solid (e.g., particulate) phase.

In the collected sample of air, interfering objects of non-interest(background) originate from the numerous, spatially variable (i.e.,varying or changing with position or location) or/and temporallyvariable (i.e., varying or changing with time) different types andconcentrations of (non-target) components (i.e., entities, materials,substances) present in the source of air. The indoor or outdoor sourceof air typically includes numerous, spatially or/and temporally variabledifferent types and concentrations of (non-target) components, such asdust (fine, dry particles of matter), pollen (fine particulate orpowderlike material consisting of pollen grains produced by plants),minerals, non-target types of biological matter (mold (fungi),bacteria), and non-target types of particulate chemical matter. Such(non-target) components in the air source can be in aerosol form, beinga gaseous suspension of fine solid or liquid particles which circulatethroughout the (indoor or outdoor) air source. Such (non-target)components have relative concentrations which, typically, spatially varyor change (i.e., vary or change with position and location) or/andtemporally vary or change (i.e., vary or change with time), dependingupon the spatial or/and temporal variations in the local atmosphericenvironment and weather conditions of the indoor or outdoor source ofair, and depending upon the location and time at which the air sample iscollected or obtained from the air source. Therefore, a plurality of airsamples is expected to have such spatially or/and temporally varying(non-target) components whose relative concentrations vary in accordancewith their spatial or/and temporal variation in the source of air fromwhich the air samples are collected or obtained.

In a similar manner, the object of interest (target), such as a(potentially hazardous) biological agent (e.g., a bacterium [such asspore-forming bacterium Bacillus anthracis], a virus, a fungus, or atoxin), or a (potentially hazardous) chemical agent (e.g., a nerve agent[e.g., sarin, tabun, or soman], or a chemical poison [e.g., a cyanidecompound, or an organophosphate compound]), which is present in a sampleof air collected or obtained from an indoor or outdoor source of air,has a relative concentration which, typically, spatially varies orchanges (i.e., varies or changes with position and location) or/andtemporally varies or changes (i.e., varies or changes with time),depending upon the spatial or/and temporal variations in the localatmospheric environment and weather conditions of the indoor or outdoorsource of air, and depending upon the location and time at which the airsample is collected or obtained from the air source. Therefore, aplurality of air samples is expected to have such a spatially or/andtemporally varying object of interest (target) whose relativeconcentration varies in accordance with its spatial or/and temporalvariation in the source of air from which the air samples are collectedor obtained.

In such an application of the present invention, typically, a givenhyper-spectrally imaged scene of a test form of an air sample includesor contains a distribution of a relatively small number of the object ofinterest (target, for example, in the form of a spectrally markedbiological or chemical agent), and a relatively large number of theobjects of non-interest (high or noisy background, in the form of(non-target) components of the air sample). Moreover, in such anapplication, typically, the majority of hyper-spectrally imaged scenesinclude or contain a relatively exceptionally small number of the objectof interest (target) compared to a relatively large number of theobjects of non-interest (background). For example, wherein the number ofthe object of interest (target), relative to the number of all objects[of interest (target) and of non-interest (background)] of (present orcontained in) a hyper-spectrally imaged scene, corresponds to a ratio orproportion as low as 1% [1 part per hundred (pph)], or 10⁻¹% [1 part perthousand (ppt)], or 10⁻⁴% [1 part per million (ppm)], 10⁻⁷% [1 part perbillion (ppb)], or even as low as 10⁻¹⁰% [1 part per trillion (pptr)].

Additionally, in such an application of the present invention, in thehyper-spectrally imaged scenes of a test form of an air sample, eachhyper-spectrally imaged object of interest (target, e.g., in the form ofa spectrally marked biological or chemical agent) and eachhyper-spectrally imaged object of non-interest (background, in the formof (non-target) components of the air sample), is definable andcharacterizable by a set of a wide variety of numerous possiblebiological, chemical, or/and physical, properties, characteristics, andbehavior. For example, in a given hyper-spectrally imaged scene, theremay occur the scenario wherein the object of interest (target, in theform of a spectrally marked biological or chemical agent) and objects ofnon-interest (background, in the form of (non-target) components of theair sample) exhibit ‘hyper-spectral’ image data and information(particularly including, for example, emission spectra corresponding tospectral representations in the form of spectral fingerprint (SFP) orsignature pattern types of identification and characterization), whichare quite similar, or even nearly identical, i.e., barelydistinguishable or resolvable, but whose ‘biological, chemical, or/andphysical’ data and information (in terms of properties, characteristics,or/and behavior), are significantly different, and not at all similar ornearly identical, i.e., not at all easily distinguishable or resolvable,or vice versa.

Regardless of the actual distributions of the different relative numbers(i.e., ratios, proportions) of the object of interest (target, in theform of a spectrally marked biological or chemical agent) and theobjects of non-interest (background, in the form of (non-target)components of the air sample), in the hyper-spectrally imaged scenes ofthe air sample, there ultimately is the need for identifying,distinguishing, and resolving, the object of interest (target, in theform of a spectrally marked biological or chemical agent) from theobjects of non-interest (background, in the form of (non-target)components of the air sample) in the hyper-spectrally imaged scenes.

This involves the need for identifying, distinguishing, and resolving,the hyper-spectral image data and information of the object of interest(target, in the form of a spectrally marked biological or chemicalagent) from that of the objects of non-interest (background, in the formof (non-target) components of the air sample). Moreover, there is alsothe need for performing such identifying, distinguishing, and resolving,procedures and operations in relation to the biological, chemical,or/and physical data and information, of the object of interest (target,in the form of a spectrally marked biological or chemical agent) and ofthe objects of non-interest (background, in the form of (non-target)components of the air sample) in the hyper-spectrally imaged scenes.Furthermore, there is also need for performing such identifying,distinguishing, and resolving, procedures and operations in view of thefact that the hyper-spectrally imaged scenes are generated and collectedfrom air samples wherein the objects of non-interest (background, in theform of (non-target) components of the air sample) and the object ofinterest (target, in the form of a spectrally marked biological orchemical agent) have relative concentrations that vary in accordancewith their spatial or/and temporal variation in the source of air fromwhich the air samples are collected or obtained.

Accordingly, the preceding described problematic and complicatingaspects, along with the corresponding negative affects and influences,due to the spatially or/and temporally varying presence of objects ofnon-interest (background) in a sample of air, make it difficult toachieve high levels of the performance parameters of accuracy, precision(reproducibility), sensitivity, resolution, or/and speed (time scale),of an overall hyper-spectral imaging and analysis application. This isparticularly the case for an exemplary specific application involvingon-line (real time or near-real time) or off-line hyper-spectral imagingand analysis of a sample of air (i.e., an air sample), for identifyingand characterizing an object of interest therein, wherein such anapplication, the interfering objects of non-interest (background) arethe numerous different spatially or/and temporally varying (non-target)components (i.e., entities, materials, substances) present in the airsample, and the object of interest (target) is a (potentially hazardous)biological agent (e.g., a bacterium [such as spore-forming bacteriumBacillus anthracis], a virus, a fungus, or a toxin), or a (potentiallyhazardous) chemical agent (e.g., a nerve agent [e.g., sarin, tabun, orsoman], or a chemical poison [e.g., a cyanide compound, or anorganophosphate compound]).

Implementation of the present invention enables overcoming the precedingdescribed problematic and complicating aspects, and correspondingnegative affects and influences, which are caused by the spatiallyor/and temporally varying presence of objects of non-interest(background) in a sample of matter, such as a sample of air, whichsignificantly limit hyper-spectral imaging and analysis of the sample ofmatter.

Preparing a Test Solution or Suspension of the Sample of Matter

In this main step (procedure), there is preparing a test solution orsuspension of the sample of matter. The test solution or suspension ofthe sample of matter is prepared in a form which is suitable for, andcompatible with, operation and use of equipment and instrumentation of agiven hyper-spectral imaging and analysis system.

Following provision of the sample of matter, or following obtaining orcollecting the sample of matter, there is preparing a test solution orsuspension of the sample of matter. Preferably, there is dissolving,suspending, or/and mixing, i.e., reformulating, a relatively smallquantity of the sample of matter into a solution or suspension form, forforming a test solution or suspension of the sample of matter. Theprocedure of dissolving, suspending, or/and mixing, i.e., reformulating,is performed using a liquid, for example, distilled water, or otherliquid, which is suitable for dissolving or suspending the differenttypes of objects of non-interest (background), i.e., (non-target)components, present in the sample of matter, and which is suitable fordissolving or suspending the object of interest (target), i.e., (target)component, present in the sample of matter.

The specific liquid used for dissolving, suspending, or/and mixing,i.e., reformulating, the small quantity of the sample of matter into atest solution or suspension, is selected whereby the liquid minimallyaffects hyper-spectral imaging and analysis of the numerous, differenttypes of objects of non-interest (background), i.e., (non-target)components, present in the sample of matter (and subsequently present inthe test solution or suspension), and whereby the liquid minimallyaffects hyper-spectral imaging and analysis of the object of interest(target), i.e., (target) component, present in the sample of matter (andsubsequently present in the test solution or suspension).

Adding to the Sample of Matter a Spectral Marker Specific to the Objectof Interest (Target)

Often, an object of interest (target), i.e., (target) component, presentin the sample of matter, by itself, is not ‘spectrally’ active, i.e.,the object of interest (target) exhibits an insufficiently detectableor/and insufficiently measurable degree or extent of luminescent (i.e.,fluorescent or/and phosphorescent) properties, characteristics, andbehavior, when illuminated by electromagnetic radiation or light, suchas ultra-violet (UV), visible (VIS), or infrared (IR), types of light.

Thus, with reference to FIG. 1, the main step (procedure) of preparing atest solution or suspension of the sample of matter includes the step(procedure) of adding to the sample of matter, a spectral markerspecific to the object of interest (target), such that if the object ofinterest (target) is present in the test solution or suspension, theobject of interest (target) when marked with the spectral marker becomesa hyper-spectrally active target which is hyper-spectrally detectableand identifiable in the test solution or suspension.

In general, the spectral marker is added to the sample of matterimmediately before or after, dissolving, suspending, or/and mixing,i.e., reformulating, the relatively small quantity of the sample ofmatter into a solution or suspension form.

The spectral marker is, in general, a chemical type of spectral marker,or a biological type of spectral marker. A suitable chemical type ofspectral marker is, in general, a chemical specie which interacts with(i.e., spectrally marks) the object of interest (target) for forming an{object of interest—chemical marker} complex which exhibits a detectableand measurable degree or extent of (spectral) luminescent (i.e.,fluorescent or/and phosphorescent) properties, characteristics, andbehavior, when illuminated by different types of electromagneticradiation or light, such as ultra-violet (UV), visible (VIS), orinfrared (IR), types of light. A suitable biological type of spectralmarker is, in general, a biological specie which interacts with (i.e.,spectrally marks) the object of interest (target) for forming an {objectof interest—biological marker} complex which exhibits some degree orextent of (spectral) luminescent (i.e., fluorescent or/andphosphorescent) properties, characteristics, and behavior, whenilluminated by different types of electromagnetic radiation or light,such as ultra-violet (UV), visible (VIS), or infrared (IR), types oflight.

In any case of the object of interest (target) being spectrally markedwith the chemical type of spectral marker, or with the biological typeof spectral marker, the formed {object of interest—chemical marker}complex, or {object of interest—biological marker} complex,respectively, becomes a hyper-spectrally active target which ishyper-spectrally detectable and identifiable in the test solution orsuspension of the sample of matter, when the test solution or suspensionis subjected to hyper-spectral imaging and analysis, thereby enablingidentifying and characterizing the object of interest in the sample ofmatter.

Adding to the Test Solution or Suspension a Background Reducing Chemical

As shown in FIG. 1, the main step (procedure) of preparing the testsolution or suspension of the sample of matter includes the unique andcritically important step (procedure) of adding to the test solution orsuspension a background reducing chemical. The background reducingchemical reduces background interfering effects caused by presence ofobjects of non-interest in the test solution or suspension, during thehyper-spectral imaging and analysis of the test solution or suspension,thereby increasing hyper-spectral detectability of the hyper-spectrallyactive target in the test solution or suspension, and thus enhancingidentification and characterization of the object of interest in thesample of matter.

The background reducing chemical is, in general, any type or kind ofchemical which is composed or made up of one or more organic or/andinorganic materials or substances, which is/are in a solid (e.g.,particulate) phase, a liquid (e.g., solution or suspension) phase, or acombination thereof.

The specific type or kind of background reducing chemical is selectedsuch that the background reducing chemical effectively (i.e.,measurably) reduces (decreases) background interfering effects caused bythe presence of the numerous, different types of objects of non-interest(background), i.e., (non-target) components, in the test solution orsuspension (particularly those objects of non-interest (background),i.e., (non-target) components, which originated from the sample ofmatter), during the hyper-spectral imaging and analysis of the testsolution or suspension. This results in increasing (enhancing)hyper-spectral detectability of the hyper-spectrally active target inthe test solution or suspension, during the hyper-spectral imaging andanalysis of the test solution or suspension. Accordingly, addition ofthe background reducing chemical to the test solution or suspension ofthe sample of matter results in increasing (enhancing) hyper-spectraldetectability of the object of interest (target), i.e., (target)component, present in the sample of matter.

The background reducing chemical, following addition to the testsolution or suspension, and during the hyper-spectral imaging of thetest solution or suspension, exhibits some combination of the followingtwo main modes of behavior.

First, the background reducing chemical selectively, ‘physicochemicallyinteracts’, during the hyper-spectral imaging, with a major portion ofthe numerous, different types of objects of non-interest (background),i.e., (non-target) components, present in the test solution orsuspension (particularly those objects of non-interest (background),i.e., (non-target) components, which originated from the sample ofmatter), in a manner which effectively (i.e., measurably) reduces(decreases) their (spectral) luminescent (i.e., fluorescent or/andphosphorescent) properties, characteristics, and behavior. During thehyper-spectral imaging, the background reducing chemical may or may noteffectively (i.e., measurably) ‘chemically react’, but at least to someextent ‘physically interacts’, with the numerous, different types ofobjects of non-interest (background), i.e., (non-target) components,present in the test solution or suspension (particularly those objectsof non-interest (background), i.e., (non-target) components, whichoriginated from the sample of matter).

Second, the background reducing chemical selectively, ‘physicochemicallyinteracts’, during the hyper-spectral imaging, with the hyper-spectrallyactive target (i.e., the spectrally marked object of interest (target),i.e., spectrally marked (target) component) present in the test solutionor suspension, in a manner which effectively (i.e., measurably)increases (enhances) its (spectral) luminescent (i.e., fluorescentor/and phosphorescent) properties, characteristics, and behavior. Duringthe hyper-spectral imaging, the background reducing chemical may or maynot effectively (i.e., measurably) ‘chemically react’, but at least tosome extent ‘physically interacts’, with the hyper-spectrally activetarget (i.e., the spectrally marked object of interest (target), i.e.,spectrally marked (target) component) present in the test solution orsuspension.

Inclusion of the step (procedure) of adding to the test solution orsuspension a background reducing chemical in the main step (procedure)of preparing a test solution or suspension of the sample of matter,results in achieving high levels of the important performance parametersof accuracy, precision (reproducibility), sensitivity, and resolution,at high speed (short time scale), be it during on-line (real time,near-real time) or off-line, in an optimum and highly efficient manner,of the remaining main steps (procedures) of the overall method, i.e.,the main step (procedure) of generating and collecting hyper-spectralimage data and information of the test solution or suspension, and themain step (procedure) of processing and analyzing the hyper-spectralimage data and information, for identifying and characterizing thehyper-spectrally active target in the test solution or suspension,thereby identifying and characterizing the object of interest in thesample of matter.

Following completion of the preceding described main step (procedure) ofpreparing a test solution or suspension of the sample of matter, aportion or aliquot of the test solution or suspension of the sample ofmatter is then, preferably, transferred and placed on a clean, inert,metal slide or plate, or, on a clean, inert, plastic (e.g., Teflon®) orglass microscope type slide or plate, which is suitable for functioningas a sample holder in a hyper-spectral imaging and analysis system. Theslide or plate (sample holder) with the portion or aliquot of the testsolution or suspension of the sample of matter is then appropriatelypositioned and secured (fixed) upon a three-dimensionally movable (i.e.,translational), and optionally, angularly movable (i.e., rotational),examination stage or platform of the hyper-spectral imaging and analysissystem. Then, there is performing the next main step (procedure) ofgenerating and collecting hyper-spectral image data and information ofthe test solution or suspension of the sample of matter.

> Exemplary Specific Embodiment for a Sample of Air (Air Sample)Containing Numerous Different Spatially or/and Temporally VaryingInterfering Objects of Non-Interest (Background) and a Biological Agentor Chemical Agent Type of Object of Interest (Target)<

Performing the preceding main step (procedure) of preparing a testsolution or suspension of the sample of matter is now described for anexemplary specific embodiment of the present invention, wherein thesample of matter is a sample of air (i.e., an air sample), and theobject of interest is a biological agent (e.g., a bacterium [such asspore-forming bacterium Bacillus anthracis], a virus, a fungus, or atoxin), or a chemical agent (e.g., a nerve agent [e.g., sarin, tabun, orsoman], or a chemical poison [e.g., a cyanide compound, or anorganophosphate compound]). In general, the object of interest (i.e.,biological agent or chemical agent) in the air sample is composed ormade up of organic or/and inorganic materials or substances, which arein a solid (e.g., particulate) phase, a liquid (e.g., solution orsuspension) phase, or/and a gaseous (e.g., aerosol) phase. Preferably,the object of interest (i.e., biological agent or chemical agent) in theair sample is composed or made up of organic or/and inorganic materialsor substances, which are in a particulate form solid phase or/and arepresent (e.g., absorbed or/and adsorbed) on particles of the air sample.More specifically, for example, the object of interest can be abiological agent, such as the spore-forming bacterium Bacillusanthracis, which is chemically marked (e.g., via terbium trichloride[TbCl₃]), or biologically marked (e.g., via antibodies of an immunoassaytechnique), as part of the main step (procedure) of preparing a testsolution or suspension of the sample of matter, i.e., the air sample,for enabling identification and characterization of the object ofinterest (i.e., the biological agent) via hyper-spectral imaging andanalysis.

The sample of air is collected or obtained (e.g., via a standard type ofair sampling or collecting system) from an indoor source of air (e.g., apost office, an airport, a subway station, a shopping mall, a sportsarena, or an office building) or from an outdoor source of air. The airsample is typically collected or obtained on a (dry or pre-wetted)porous (filter-like or screen-like) solid substrate, such as a plasticor fiberglass (filter-like or screen-like) substrate, which functionslike a filter, for filtering, capturing, and collecting a sample fromthe source of air.

The particulate matter of the air sample which is collected on theporous (filter-like or screen-like) solid substrate typically includesnumerous, different types and concentrations of objects of non-interest,i.e., (non-target) components, such as dust, pollen, minerals,non-target types of biological matter (mold (fungi), bacteria), andnon-target types of particulate chemical matter. Such (non-target)components of the air sample have relative concentrations which,typically, spatially vary or change (i.e., vary or change with positionand location) or/and temporally vary or change (i.e., vary or changewith time), depending upon the spatial or/and temporal variations in thelocal atmospheric environment and weather conditions of the indoor oroutdoor source of air, and depending upon the location and time at whichthe air sample is collected or obtained from the air source. Therefore,a plurality of air samples is expected to have such spatially or/andtemporally varying (non-target) components whose relative concentrationsvary in accordance with their spatial or/and temporal variation in thesource of air from which the air samples are collected or obtained.

The air sample which is collected on the porous (filter-like orscreen-like) solid substrate may also include an object of interest(target), i.e., (target) component, such as a biological agent (e.g., abacterium [such as spore-forming bacterium Bacillus anthracis], a virus,a fungus, or a toxin), or a chemical agent (e.g., a nerve agent [e.g.,sarin, tabun, or soman], or a chemical poison [e.g., a cyanide compound,or an organophosphate compound]). Such a (target) component of the airsample has a relative concentration which, typically, spatially variesor changes (i.e., varies or changes with position and location) or/andtemporally varies or changes (i.e., varies or changes with time),depending upon the spatial or/and temporal variations in the localatmospheric environment and weather conditions of the indoor or outdoorsource of air, and depending upon the location and time at which the airsample is collected or obtained from the air source. Therefore, aplurality of air samples is expected to have such a spatially or/andtemporally varying object of interest (target) whose relativeconcentration varies in accordance with its spatial or/and temporalvariation in the source of air from which the air samples are collectedor obtained.

Thus, following provision of the air sample, or following obtaining orcollecting the air sample, there is preparing an appropriate test formof the air sample.

Utilizing the porous (filter-like or screen-like) solid substrate uponwhich is the collected particulate matter of the air sample, preferably,there is dissolving or suspending a relatively small quantity of the airsample into a solution or suspension form, for forming a test solutionor suspension of the air sample. The procedure of dissolving,suspending, or/and mixing, i.e., reformulating, is performed using aliquid, for example, distilled water, or other liquid, which is suitablefor dissolving or suspending the different types of objects ofnon-interest, i.e., (non-target) components present in the air sample(i.e., dust, pollen, minerals, non-target types of biological matter(mold (fungi), bacteria), and non-target types of particulate chemicalmatter), and which is suitable for dissolving or suspending the objectof interest (target), such as a biological agent (e.g., a bacterium[such as spore-forming bacterium Bacillus anthracis], a virus, a fungus,or a toxin), or a chemical agent (e.g., nerve agent [e.g., sarin, tabun,or soman], or a chemical poison [e.g., a cyanide compound, or anorganophosphate compound]).

The specific liquid used for dissolving, suspending, or/and mixing,i.e., reformulating, the small quantity of the air sample into asolution or suspension form, for forming a test solution or suspensiontest of the air sample, is selected whereby the liquid minimally affectshyper-spectral imaging and analysis of the numerous, different types ofobjects of non-interest, i.e., (non-target) components (i.e., dust,pollen, minerals, non-target types of biological matter (mold (fungi),bacteria), and non-target types of particulate chemical matter), presentin the air sample (and subsequently present in the test solution orsuspension), and whereby the liquid minimally affects hyper-spectralimaging and analysis of the object of interest (target), i.e., (target)component, such as a biological agent (e.g., a bacterium [such asspore-forming bacterium Bacillus anthracis], a virus, a fungus, or atoxin), or a chemical agent (e.g., a nerve agent [e.g., sarin, tabun, orsoman], or a chemical poison [e.g., a cyanide compound, or anorganophosphate compound]), present in the air sample (and subsequentlypresent in the test solution or suspension).

Adding to the Air Sample a Spectral Marker Specific to the BiologicalAgent or Chemical Agent

Often, the object of interest (target), such as a biological agent(e.g., a bacterium [such as spore-forming bacterium Bacillus anthracis],a virus, a fungus, or a toxin), or a chemical agent (e.g., a nerve agent[e.g., sarin, tabun, or soman], or a chemical poison [e.g., a cyanidecompound, or an organophosphate compound]), by itself, is not‘spectrally’ active, i.e., the biological agent or chemical agent(target) exhibits an insufficiently detectable or/and insufficientlymeasurable degree or extent of luminescent (i.e., fluorescent or/andphosphorescent) properties, characteristics, and behavior, whenilluminated by electromagnetic radiation or light, such as ultra-violet(UV), visible (VIS), or infrared (IR), types of light.

Thus, the main step (procedure) of preparing a test solution orsuspension of the air sample includes the step (procedure) of adding tothe air sample, a spectral marker specific to the (target) biologicalagent or chemical agent, such that if the (target) biological agent orchemical agent is present in the test solution or suspension, the(target) biological agent or chemical agent when marked with thespectral marker becomes a hyper-spectrally active target which ishyper-spectrally detectable and identifiable in the test solution orsuspension.

The spectral marker is, in general, a chemical type of spectral marker,or a biological type of spectral marker. A suitable chemical type ofspectral marker is, in general, a chemical specie which interacts with(i.e., spectrally marks) the (target) biological agent or chemical agentfor forming a {biological agent-chemical marker} complex or a {chemicalagent—chemical marker} complex, respectively, which exhibits adetectable and measurable degree or extent of luminescent (i.e.,fluorescent or/and phosphorescent) properties, characteristics, andbehavior, when illuminated by different types of electromagneticradiation or light, such as ultra-violet (UV), visible (VIS), orinfrared (IR), types of light. A suitable biological type of spectralmarker is, in general, a biological specie which interacts with (i.e.,spectrally marks) the (target) biological agent or chemical agent forforming a {biological agent-biological marker} complex or a {chemicalagent—biological marker} complex, respectively, which exhibits somedegree or extent of luminescent (i.e., fluorescent or/andphosphorescent) properties, characteristics, and behavior, whenilluminated by different types of electromagnetic radiation or light,such as ultra-violet (UV), visible (VIS), or infrared (IR), types oflight.

In any case of the (target) biological agent or chemical agent beingspectrally marked with the chemical type of spectral marker, or with thebiological type of spectral marker, the formed {biologicalagent-chemical marker} complex or {chemical agent—chemical marker}complex, respectively, or, the formed {biological agent-biologicalmarker} complex or {chemical agent-biological marker} complex,respectively, becomes a hyper-spectrally active target which ishyper-spectrally detectable and identifiable in the test solution orsuspension of the air sample, when the test solution or suspension issubjected to hyper-spectral imaging and analysis, thereby enablingidentifying and characterizing the object of interest (i.e., biologicalagent or chemical agent) in the sample of matter.

Specific Case of the Biological Agent (e.g., Bacterium, Virus, Fungus,or Toxin) being the Spore-Forming Bacterium Bacillus anthracis

In a specific case of the exemplary specific embodiment of the presentinvention, wherein the sample of matter is a sample of air (i.e., an airsample), and the object of interest is a biological agent (e.g.,bacterium, virus, fungus, or toxin), such as the spore-forming bacteriumBacillus anthracis, then, this main step (procedure) is performed asfollows.

There is adding to the air sample, a spectral marker, specific to the(target) spore-forming bacterium Bacillus anthracis, such that if the(target) spore-forming bacterium Bacillus anthracis is present in thetest solution or suspension, the (target) spore-forming bacteriumBacillus anthracis when marked with the spectral marker becomes ahyper-spectrally active target which is hyper-spectrally detectable andidentifiable in the test solution or suspension.

In general, the spectral marker is added to the air sample immediatelybefore or after, dissolving, suspending, or/and mixing, i.e.,reformulating, the relatively small quantity of the air sample into asolution or suspension form.

The spectral marker is, in general, a chemical type of spectral marker,or a biological type of spectral marker. A suitable chemical type ofspectral marker is a chemical specie, for example, terbium trichloride[TbCl₃], which interacts with (i.e., spectrally marks) the (target)spore-forming bacterium Bacillus anthracis (via dipicolinic acid [DPA]therein) for forming a {biological agent Bacillus anthracis spore(dipicolinic acid [DPA])—terbium trichloride [TbCl₃]} complex, whichexhibits a detectable and measurable degree or extent of luminescent(i.e., fluorescent or/and phosphorescent) properties, characteristics,and behavior, when illuminated by different types of electromagneticradiation or light, such as ultra-violet (UV), visible (VIS), orinfrared (IR), types of light. A suitable biological type of spectralmarker is a biological specie, for example, an antibody of animmunoassay technique (for example, as described in reference 37), whichinteracts with (i.e., spectrally marks) the (target) spore-formingbacterium Bacillus anthracis (via surface antigens thereof) for forminga {biological agent Bacillus anthracis spore antigen—antibody} complex,which exhibits a detectable and measurable degree or extent ofluminescent (i.e., fluorescent or/and phosphorescent) properties,characteristics, and behavior, when illuminated by different types ofelectromagnetic radiation or light, such as ultra-violet (UV), visible(VIS), or infrared (IR), types of light.

In any case of the (target) spore-forming bacterium Bacillus anthracisbeing spectrally marked with the chemical type of spectral marker (i.e.,terbium trichloride [TbCl₃]), or with the biological type of spectralmarker (i.e., antibody), the formed {biological agent Bacillus anthracisspore (dipicolinic acid [DPA])—terbium trichloride [TbCl₃]} complex, or{biological agent Bacillus anthracis spore antigen—antibody} complex,respectively, becomes a hyper-spectrally active target which ishyper-spectrally detectable and identifiable in the test solution orsuspension of the air sample, when the test solution or suspension issubjected to hyper-spectral imaging and analysis.

Specific Case of Adding to the Test Solution or Suspension a BackgroundReducing Chemical, For Example, an Organic Liquid, Such as EthyleneGlycol (Meg) [HOCH₂CH₂OH]

In accordance with the method of the present invention, as shown in FIG.1, the main step (procedure) of preparing the test solution orsuspension of the air sample includes the unique and criticallyimportant step (procedure) of adding to the test solution or suspensiona background reducing chemical. The background reducing chemical reducesbackground interfering effects caused by presence of numerous, differenttypes of objects of non-interest (background), i.e., (non-target)components, in the test solution or suspension (particularly thoseobjects of non-interest (background), i.e., (non-target) components,which originated from the air sample), during the hyper-spectral imagingand analysis of the test solution or suspension, thereby increasinghyper-spectral detectability of the hyper-spectrally active target(i.e., the {biological agent Bacillus anthracis spore (dipicolinic acid[DPA])—terbium trichloride [TbCl₃]} complex, or the {biological agentBacillus anthracis spore antigen—antibody} complex) in the test solutionor suspension, and thus enhancing identification and characterization ofthe object of interest (i.e., the Bacillus anthracis biological agent)in the sample of matter.

The background reducing chemical is, in general, any type or kind ofchemical which is composed or made up of one or more organic or/andinorganic materials or substances, which is/are in a solid (e.g.,particulate) phase, a liquid (e.g., solution or suspension) phase, or acombination thereof.

In the specific case of the exemplary specific embodiment of the presentinvention, wherein the sample of matter is a sample of air (i.e., an airsample), and the object of interest is a biological agent (e.g.,bacterium, virus, fungus, or toxin), such as the spore-forming bacteriumBacillus anthracis, it was empirically determined (see the Exampleshereinbelow) that an exemplary preferred background reducing chemical isan organic liquid, such as ethylene glycol (i.e., monoethylene glycol(MEG) or ethane-1,2-diol) [HOCH₂CH₂OH].

The specific type or kind of background reducing chemical, i.e., theethylene glycol (MEG) [HOCH₂CH₂OH], is selected such that the backgroundreducing chemical effectively (i.e., measurably) reduces (decreases)background interfering effects caused by the presence of the numerous,different types of objects of non-interest (background), i.e.,(non-target) components, in the test solution or suspension(particularly the numerous, spatially or/and temporally variabledifferent types and concentrations of (non-target) components, such asdust, pollen, minerals, non-target types of biological matter (mold(fungi), bacteria), and non-target types of particulate chemical matter,which originated from the air sample), during the hyper-spectral imagingand analysis of the test solution or suspension. This results inincreasing (enhancing) hyper-spectral detectability of thehyper-spectrally active target (i.e., the {biological agent Bacillusanthracis spore (dipicolinic acid [DPA])—terbium trichloride [TbCl₃]}complex, or the {biological agent Bacillus anthracis sporeantigen—antibody} complex) in the test solution or suspension, duringthe hyper-spectral imaging and analysis of the test solution orsuspension. Accordingly, addition of the background reducing chemical,i.e., the ethylene glycol (MEG) [HOCH₂CH₂OH], to the test solution orsuspension of the air sample results in increasing (enhancing)hyper-spectral detectability of the (target) spore-forming bacteriumBacillus anthracis, present in the air sample.

The background reducing chemical, i.e., the ethylene glycol (MEG)[HOCH₂CH₂OH], following addition to the test solution or suspension, andduring the hyper-spectral imaging of the test solution or suspension,exhibits some combination of the following two main modes of behavior.

First, the background reducing chemical, i.e., the ethylene glycol (MEG)[HOCH₂CH₂OH], selectively, ‘physicochemically interacts’, during thehyper-spectral imaging, with a major portion of the numerous, differenttypes of objects of non-interest (background), i.e., (non-target)components, present in the test solution or suspension (particularlythose objects of non-interest (background), i.e., (non-target)components, which originated from the air sample), in a manner whicheffectively (i.e., measurably) reduces (decreases) their (spectral)luminescent (i.e., fluorescent or/and phosphorescent) properties,characteristics, and behavior. During the hyper-spectral imaging, thebackground reducing chemical, i.e., the ethylene glycol (MEG)[HOCH₂CH₂OH], may or may not effectively (i.e., measurably) ‘chemicallyreact’, but at least to some extent ‘physically interacts’, with thenumerous, different types of objects of non-interest (background), i.e.,(non-target) components, present in the test solution or suspension(particularly including those objects of non-interest (background),i.e., (non-target) components, which originated from the air sample).

Second, the background reducing chemical, i.e., the ethylene glycol(MEG) [HOCH₂CH₂OH], selectively, ‘physicochemically interacts’, duringthe hyper-spectral imaging, with the hyper-spectrally active target(i.e., the {biological agent Bacillus anthracis spore (dipicolinic acid[DPA])—terbium trichloride [TbCl₃]} complex, or the {biological agentBacillus anthracis spore antigen—antibody} complex) present in the testsolution or suspension, in a manner which effectively (i.e., measurably)increases (enhances) its (spectral) luminescent (i.e., fluorescentor/and phosphorescent) properties, characteristics, and behavior. Duringthe hyper-spectral imaging, the background reducing chemical, i.e., theethylene glycol (MEG) [HOCH₂CH₂OH], may or may not effectively (i.e.,measurably) ‘chemically react’, but at least to some extent ‘physicallyinteracts’, with the hyper-spectrally active target (i.e., the{biological agent Bacillus anthracis spore (dipicolinic acid[DPA])—terbium trichloride [TbCl₃]} complex, or the {biological agentBacillus anthracis spore antigen—antibody} complex) present in the testsolution or suspension.

Inclusion of the step (procedure) of adding to the test solution orsuspension a background reducing chemical, i.e., the ethylene glycol(MEG) [HOCH₂CH₂OH], in the main step (procedure) of preparing a testsolution or suspension of the air sample, results in achieving highlevels of the performance parameters of accuracy, precision(reproducibility), sensitivity, at high speed (short time scale), be itduring on-line (real time, near-real time) or off-line, in an optimumand highly efficient manner, of the remaining main steps (procedures) ofthe method, i.e., the main step (procedure) of generating and collectinghyper-spectral image data and information of the test solution orsuspension, and the main step (procedure) of processing and analyzingthe hyper-spectral image data and information, for identifying andcharacterizing the hyper-spectrally active target (i.e., the {biologicalagent Bacillus anthracis spore (dipicolinic acid [DPA])—terbiumtrichloride [TbCl₃]} complex, or the {biological agent Bacillusanthracis spore antigen—antibody} complex) in the test solution orsuspension, thereby identifying and characterizing the object ofinterest (i.e., the spore-forming bacterium Bacillus anthracis) in theair sample.

Following completion of the preceding described main step (procedure) ofpreparing a test solution or suspension of the air sample, a portion oraliquot of the test solution or suspension of the air sample is then,preferably, transferred and placed on a clean, inert, metal slide orplate, or, on a clean, inert, plastic (e.g., Teflon®) or glassmicroscope type slide or plate, which is suitable for functioning as asample holder in a hyper-spectral imaging and analysis system. The slideor plate (sample holder) with the portion or aliquot of the testsolution or suspension of the air sample is then appropriatelypositioned and secured (fixed) upon a three-dimensionally movable (i.e.,translational), and optionally, angularly movable (i.e., rotational),examination stage or platform of the hyper-spectral imaging and analysissystem. Then, there is performing the next main step (procedure) ofgenerating and collecting hyper-spectral image data and information ofthe test solution or suspension of the air sample.

Generating and Collecting Hyper-Spectral Image Data and Information ofthe Test Solution or Suspension

This main step (procedure) of the method for hyper-spectral imaging andanalysis of a sample of matter, for identifying and characterizing anobject of interest therein, of the present invention, is performed asdescribed in the Field and Background section, hereinabove. In general,this main step (procedure) of generating and collecting hyper-spectralimage data and information of the test solution or suspension of thesample of matter, is performed according to any suitable teaching orpractice of generating and collecting hyper-spectral image data andinformation, using any suitable hyper-spectral imaging and analysissystem and technique.

For example, for performing this main step (procedure) of the method ofthe present invention, there is using any suitable teaching or practicedisclosed in references 1-29 (and references cited therein). Preferably,there is using the selected teachings and practices of hyper-spectralimaging and analysis by the same applicant/assignee of the presentinvention which are disclosed in references 30-36.

Accordingly, from the preceding described main step (procedure) of themethod, the slide or plate (sample holder) with the portion or aliquotof the test solution or suspension of the sample of matter which isappropriately positioned and secured (fixed) upon a three-dimensionallymovable (i.e., translational), and optionally, angularly movable (i.e.,rotational), examination stage or platform of the hyper-spectral imagingand analysis system, is subjected to subjected to hyper-spectral imagingand analysis. Multiple fields of view of the sample of matter are‘hyper-spectrally’ scanned and imaged while the sample of matter(containing objects, and components thereof) is exposed toelectromagnetic radiation. During the hyper-spectral scanning andimaging there is generating and collecting relatively large numbers (upto the order of millions) of multiple spectral (i.e., hyper-spectral)images, ‘one-at-a-time’, but, in an extremely fast or rapid sequentialmanner, of the objects (and components thereof) emitting electromagneticradiation at a plurality of many wavelengths and frequencies, where thewavelengths and frequencies are associated with different selected(relatively narrow) portions or bands, or bands therein, of an entirehyper-spectrum emitted by the objects (and components thereof) of thesample of matter. The hyper-spectral imaging and analysis system can beoperated in an extremely fast or rapid manner for providingexceptionally highly resolved spectral and spatial data and informationof the imaged sample of matter (containing the objects, and componentsthereof).

>Exemplary Specific Embodiment for a Sample of Air (Air Sample)Containing Numerous Different Spatially or/and Temporally VaryingInterfering Objects of Non-Interest (Background), and a Biological Agentor Chemical Agent Type of Object of Interest (Target)<

This main step (procedure) is readily performed for generating andcollecting hyper-spectral image data and information of the testsolution or suspension, for the exemplary specific embodiment of thepresent invention, wherein the sample of matter is a sample of air(i.e., an air sample), and the object of interest is a biological agent(e.g., a bacterium, a virus, a fungus, or a toxin), or a chemical agent(e.g., a nerve agent [e.g., sarin, tabun, or soman], or a chemicalpoison [e.g., a cyanide compound, or an organophosphate compound]),which is composed or made up of organic or/and inorganic materials orsubstances that are preferably in a solid (e.g., particulate) phaseor/and are present (e.g., absorbed or/and adsorbed) on particles of theair sample. More specifically, for example, the object of interest canbe a biological agent, such as the spore-forming bacterium Bacillusanthracis, which is chemically marked (e.g., via terbium trichloride[TbCl₃]), or biologically marked (e.g., via antibodies of an immunoassaytechnique), as described hereinabove, as part of the main step(procedure) of preparing a test solution or suspension of the sample ofmatter, i.e., the air sample, for enabling identification andcharacterization of the objects of interest (i.e., the biological agentspore-forming bacterium Bacillus anthracis) via hyper-spectral imagingand analysis.

Processing and Analyzing the Hyper-Spectral Image Data and Information

This main step (procedure) of the method for hyper-spectral imaging andanalysis of a sample of matter, for identifying and characterizing anobject of interest therein, of the present invention, is performed asdescribed in the Field and Background section, hereinabove. In general,this main step (procedure) of processing and analyzing thehyper-spectral image data and information, for identifying andcharacterizing the hyper-spectrally active target in the test solutionor suspension, thereby identifying and characterizing the object ofinterest in the sample of matter, is performed according to any suitableprior art teaching or practice of processing and analyzinghyper-spectral image data and information, for identifying andcharacterizing a hyper-spectrally active target in a test solution orsuspension, using any suitable hyper-spectral imaging and analysissystem taught about or practiced in the prior art.

For example, for performing this main step (procedure) of the method ofthe present invention, there is using any suitable teaching or practicedisclosed in references 1-29 (and references cited therein). Preferably,there is using the selected teachings and practices of hyper-spectralimaging and analysis by the same applicant/assignee of the presentinvention which are disclosed in references 30-36.

Accordingly, the hyper-spectral images generated by, and collected from,the sample of matter, are correlated with emission spectra of the sampleof matter, where the emission spectra correspond to spectralrepresentations in the form of spectral ‘fingerprint’ or ‘signature’pattern types of identification and characterization, of thehyper-spectrally imaged objects (and components thereof) in the sampleof matter. Such hyper-spectral image data and information are processedand analyzed by using automatic pattern recognition (APR) or/and opticalcharacter recognition (OCR) types of hyper-spectral imaging data andinformation processing and analysis, for identifying, characterizing,or/and classifying, the physical, chemical, or/and biological,properties, characteristics, and behavior, of the hyper-spectrallyimaged objects (and components thereof) in the sample of matter.

>Exemplary Specific Embodiment for a Sample of Air (Air Sample)Containing Numerous Different Spatially or/and Temporally VaryingInterfering Objects of Non-Interest (Background), and a Biological AgentOr Chemical Agent Type Of Object Of Interest (Target)<

This main step (procedure) is readily performed for processing andanalyzing the hyper-spectral image data and information, for identifyingand characterizing the hyper-spectrally active target in the testsolution or suspension, thereby identifying and characterizing theobject of interest in the sample of matter, for the exemplary specificpreferred embodiment of the present invention, wherein the sample ofmatter is a sample of air (i.e., an air sample), and the object ofinterest is a biological agent (e.g., a bacterium, a virus, a fungus, ora toxin), or a chemical agent (e.g., a nerve agent [e.g., sarin, tabun,or soman], or a chemical poison [e.g., a cyanide compound, or anorganophosphate compound]). More specifically, for example, the objectof interest can be a biological agent, such as the spore-formingbacterium Bacillus anthracis, which is chemically marked (e.g., viaterbium trichloride [TbCl₃]), or biologically marked (e.g., viaantibodies of an immunoassay technique), as described hereinabove, aspart of the main step (procedure) of preparing a test solution orsuspension of the sample of matter, i.e., the air sample, for enablingidentification and characterization of the objects of interest (i.e.,the biological agent spore-forming bacterium Bacillus anthracis) viahyper-spectral imaging and analysis.

According to the second main aspect of the present invention, there isprovision of a method for preparing a test solution or suspension from asample of matter, the test solution or suspension being particularlysuitable for subjecting to hyper-spectral imaging and analysis.

Referring to the first (or top) box in the flow diagram drawn in FIG. 1,the method for preparing a test solution or suspension from a sample ofmatter includes the following main steps or procedures, and, componentsand functionalities thereof: preparing a solution or suspension of thesample of matter, the preparing includes adding to the sample of mattera spectral marker specific to an object of interest, such that if theobject of interest is present in the test solution or suspension, theobject of interest when marked with the spectral marker becomes ahyper-spectrally active target which is hyper-spectrally detectable andidentifiable in the test solution or suspension; characterized in thatpreparing the test solution or suspension includes adding to thesolution or suspension a background reducing chemical, thereby formingthe test solution or suspension, wherein the background reducingchemical reduces background interfering effects caused by presence ofobjects of non-interest in the test solution or suspension, duringhyper-spectral imaging and analysis of the test solution or suspension,thereby increasing hyper-spectral detectability of the hyper-spectrallyactive target in the test solution or suspension.

The method for preparing a test solution or suspension from a sample ofmatter, the test solution or suspension being particularly suitable forsubjecting to hyper-spectral imaging and analysis, of the presentinvention, is applicable to essentially any type or kind, and form, ofsample of matter, in the same manner as described hereinabove withrespect to the method for hyper-spectral imaging and analysis of asample of matter, for identifying and characterizing an object ofinterest therein, of the present invention. Additionally, in the methodfor preparing a test solution or suspension from a sample of matter, ingeneral, in the sample of matter (including the objects and componentsthereof present in the sample of matter), the objects (i.e., entities,materials, substances) are typed, categorized, or classified, accordingto two main different types, categories, or classes, namely, ‘objects ofnon-interest’, and ‘objects of interest’, in the same manner asdescribed hereinabove with respect to the method for hyper-spectralimaging and analysis of a sample of matter.

The method for preparing a test solution or suspension from a sample ofmatter, the test solution or suspension being particularly suitable forsubjecting to hyper-spectral imaging and analysis, of the presentinvention, is performed in the same manner as, and in full accordancewith, the hereinabove described main step (procedure) of preparing atest solution or suspension of the sample of matter, of the method forhyper-spectral imaging and analysis of a sample of matter, foridentifying and characterizing an object of interest therein, of thepresent invention.

>Exemplary Specific Embodiment for a Sample of Air (Air Sample)Containing Numerous Different Spatially or/and Temporally VaryingInterfering Objects of Non-Interest (Background), and a Biological Agentor Chemical Agent Type of Object of Interest (Target)<

In the same manner as, and in full accordance with, the hereinabovedescribed main step (procedure) of preparing a test solution orsuspension of the sample of matter, of the method for hyper-spectralimaging and analysis of a sample of matter, for identifying andcharacterizing an object of interest therein, of the present invention,herein, the method for preparing a test solution or suspension from asample of matter, the test solution or suspension being particularlysuitable for subjecting to hyper-spectral imaging and analysis, isreadily performed for the exemplary specific embodiment of the presentinvention, wherein the sample of matter is a sample of air (i.e., an airsample), and the object of interest is a biological agent (e.g., abacterium, a virus, a fungus, or a toxin), or a chemical agent (e.g., anerve agent [e.g., sarin, tabun, or soman], or chemical poison [e.g., acyanide compound, or an organophosphate compound]).

In general, the object of interest (i.e., biological agent or chemicalagent) in the air sample is composed or made up of organic or/andinorganic materials or substances, which are in a solid (e.g.,particulate) phase, a liquid (e.g., solution or suspension) phase,or/and a gaseous (e.g., aerosol) phase. Preferably, the object ofinterest (i.e., biological agent or chemical agent) in the air sample iscomposed or made up of organic or/and inorganic materials or substances,which are in a particulate form solid phase or/and are present (e.g.,absorbed or/and adsorbed) on particles of the air sample. Morespecifically, for example, the object of interest can be a biologicalagent, such as the spore-forming bacterium Bacillus anthracis, which ischemically marked (e.g., via terbium trichloride [TbCl₃]), orbiologically marked (e.g., via antibodies of an immunoassay technique),as described hereinabove, as part of the main step (procedure) ofpreparing a test solution or suspension of the sample of matter, i.e.,the air sample, for enabling identification and characterization of theobject of interest (i.e., the spore-forming bacterium Bacillusanthracis) via hyper-spectral imaging and analysis.

EXAMPLES

Selected embodiments of the present invention, including novel andinventive aspects, characteristics, special technical features, andadvantages thereof, as illustratively described hereinabove, and asclaimed in the claims section hereinbelow, are exemplified and haveexperimental support in the following examples, which are not intendedto be limiting.

Materials and Procedures Hyper-Spectral Imaging and Analysis System

In each of the Examples 1-3, the same hyper-spectral imaging andanalysis system was used: the Green Vision Systems Ltd. (Tel Aviv,Israel) manufactured hyper-spectral imaging (optical scanning) andanalysis system, model names: HyperEye® Detection System, FIPA®(Fluorescence Imaging Particle Analyzer), and FIPA-20® (FluorescenceImaging Particle Analyzer), including the following main components andoperational features or conditions.

Sample illuminating (light beam) source: mercury lamp, 100 W (watts),with a narrow band pass filter centered at 334 or 365 nanometers (nm)and bandwidth of ±10 mm.Optical scan bandwidth: at or within the range of 400-900 mm.Digital analysis range: 400-900 nm.Optics: objective, ×10 Zeiss Ultrafluar®, with a pixel size of 6.45microns (μ)×6.45μ.Wavelength resolution: 1.5 nm.

The hyper-spectral imaging and analysis system included athree-dimensionally movable (i.e., translational), and angularly movable(i.e., rotational), examination stage or platform, upon which wereplaced test samples.

Chemical and Biological Reagents

Water (Examples 1-3, for washing and rinsing): distilled deionizedfiltered water, generated by a research laboratory grade waterpurification system, was exclusively used throughout.Ethanol (Examples 2, 3): dehydrated ethyl alcohol, >85.0%, Sigma-Aldrich(no. 636-1).Ethylene glycol (Examples 2 and 3): ethylene glycol (monoethylene glycol(MEG) or ethane-1,2-diol) [HOCH₂CH₂OH], ≧98.0%, Sigma-Aldrich (Flukabrand, no. 03760).Dipicolinic acid [DPA] (Example 3): 2,6-pyridinedicarboxylic acid, 99%,Sigma-Aldrich (Aldrich brand, no. P63808).Terbium trichloride [TbCl₃] (Example 3): terbium (III) chloride,anhydrous powder, 99% (metal basis), Sigma-Aldrich (Aldrich brand, no.439657).Biological agent, Bacillus subtilis spores (Examples 3 and 4): Bacillussubtilis (ATCC 6633), lyophilized cells, Sigma-Aldrich (Sigma brand, no.B4006). The (non-hazardous) biological agent Bacillus subtilis was usedas a functional equivalent substitute for the (extremely hazardous)biological agent Bacillus anthracis.

General Experimental Methods and Procedures

Examples 1-3 are ‘actual’ or ‘working’ examples, wherein the describedand illustrated experimental methodologies and procedures were developedand performed, and the experimental results were obtained, by the sameapplicant/assignee of the present invention.

In Example 3, preparing test solutions or suspensions of the indicatedsamples of matter were performed according to the hereinabovedescription of the present invention.

In Examples 1-3, generating and collecting, and, processing andanalyzing, the hyper-spectral image data and information of theindicated samples of matter were performed according to the hereinabovedescription of the present invention, according to the selectedteachings and practices of hyper-spectral imaging and analysis by thesame applicant/assignee of the present invention which are disclosed inreferences 30-36.

Example 4 is ‘prophetic’, wherein the (prophetic) experimentalmethodology and procedures were developed by the same applicant/assigneeof the present invention.

Example 1 Hyper-Spectral Imaging and Analysis of ‘Dust Particles’Present in a Sample of Air Collected from an Indoor Source of Air

In Example 1, (dry form) dust particles of different sizes and shapespresent in samples of air (i.e., air samples) collected from an indoorsource of air (absent of any particular object of interest (target),such as a biological agent or chemical agent) were subjected tohyper-spectral imaging and analysis. The main objective was forobtaining hyper-spectral image data and information in the form ofimages and spectral fingerprint (SFP) or spectral signature patterntypes of identification and characterization of the various differentsized and shaped dust particles present in the source of air, forserving as a useful reference associated with ‘objects of non-interest’(background) during hyper-spectrally imaging and analyzing of similarlycollected air samples, or of other types of samples of matter, which mayhave present an ‘object of interest’ (target), such as a biologicalagent or chemical agent.

Experimental Methods and Procedures

Samples of air (air samples) were collected, using a standard type ofair sampling or collecting system, from an indoor source of air (absentof any particular object of interest (target), such as a biologicalagent or chemical agent), being air in the immediate vicinity wherepostal workers handle and process letters inside a post office. The postoffice normally had closed windows, with a standard type of HVAC(heating, ventilation, air conditioning) environmental control. Each airsample was collected on a (dry or pre-wetted) porous plastic orfiberglass (filter-like or screen-like) substrate, which functioned likea filter, for filtering, capturing, and collecting, a sample from thesource of air.

The indoor source of air typically included numerous, spatially or/andtemporally variable different types and concentrations of components,such as dust (fine, dry particles of matter), pollen (fine particulateor powderlike material consisting of pollen grains produced by plants),minerals, non-target types of biological matter (mold (fungi),bacteria), and non-target types of particulate chemical matter. All suchcomponents present in the collected air samples were composed or made upof organic or/and inorganic materials or substances, which were in aparticulate form solid phase or/and were present (e.g., absorbed or/andadsorbed) on particles of the collected air samples, and werecollectively classified and treated as ‘dust particles’. The dustparticles had a characteristic size in the range of between about 1micron and about 20 microns, and were of different shapes.

For each air sample, a portion of the (dry form) dust particles wasplaced (spread out) on a clean, inert, glass microscope type slide orplate which functioned as a sample holder in the hyper-spectral imagingand analysis system. The slide or plate (sample holder) with the portionof the dust particles was then appropriately positioned and secured(fixed) upon the examination stage or platform of the hyper-spectralimaging and analysis system. Fifty separate (individual) dust particles,of different sizes and shapes, were hyper-spectrally scanned and imaged,for obtaining hyper-spectral images and spectral fingerprint (SFP) orspectral signature pattern types of identification and characterization.

Results

The results for two exemplary and representative separate (individual)dust particles of the fifty separate (individual) dust particles whichwere subjected to the hyper-spectral imaging and analysis are presentedin FIGS. 2 a and 2 b, which are exemplary empirically determinedgraphical plots (spectra) of Emission Intensity (normalized) as afunction of Emission Wavelength (nm) of exemplary dust particles 1 and2, respectively, showing the spectral fingerprints (SFPs) thereof. FIGS.2 a and 2 b show that each of dust particle 1 and dust particle 2,respectively, is characterized by essentially the same spectralfingerprint (SFP) having essentially the same characteristic shape, andsame characteristic emission intensity maximum or peak at an emissionwavelength of about 460 nm.

Example 2 Hyper-Spectral in Aging and Analysis of (Native) ‘DustParticles’ Suspended in Different Liquids (Ethanol, or Ethylene Glycol),for Determining Extent of ‘Background’ Signal Reduction Effected by EachLiquid

In Example 2, (native) dust particles of different sizes and shapesmanually collected from dust originating from an indoor source of air(absent of any particular object of interest (target), such as abiological agent or chemical agent) were suspended in different liquids(ethanol, or ethylene glycol) and subjected to hyper-spectral imagingand analysis. The main objective was for obtaining hyper-spectral imagedata and information in the form of hyper-spectral images and spectralfingerprint (SFP) or spectral signature pattern types of identificationand characterization of the suspended dust particles (as exemplary testsuspensions), for determining the extent or degree of ‘background’signal reduction that could be effected by each suspending liquid. Theexperimental data and information serves as useful reference associatedwith ‘objects of non-interest’ (background) during hyper-spectrallyimaging and analyzing of air samples, or of other types of samples ofmatter, which may have present an ‘object of interest’ (target), such asa biological agent or chemical agent.

Experimental Methods and Procedures

(Native) dust particles were manually collected (using inert ‘dust free’surgical type clean gloves and a metal spatula) from a normally clean,‘dusty’ desktop. The dust particles originated from an indoor source ofair (absent of any particular object of interest, such as a biologicalagent or chemical agent), being air of a business office which normallyhad closed windows, with a standard type of HVAC (heating, ventilation,air conditioning) environmental control.

As for Example 1, the indoor source of air typically included numerous,spatially or/and temporally variable different types and concentrationsof components, such as dust, pollen, minerals, non-target types ofbiological matter, and non-target types of particulate chemical matter.All such components present in the collected dust samples were composedor made up of organic or/and inorganic materials or substances, whichwere in a particulate form solid phase or/and were present (e.g.,absorbed or/and adsorbed) on particles of the indoor air, and werecollectively classified and treated as ‘dust particles’. The dustparticles had a characteristic size in the range of between about 1micron and about 20 microns, and were of different shapes.

A series of three types of samples (sample types) were prepared andtested: (Type 1 samples): dry form dust particles (as a baselinereference or control) absent of any liquid; (Type 2 samples): a testsuspension of dust particles suspended in ethanol; and (Type 3 samples):a test suspension of dust particles suspended in ethylene glycol.

For each sample type (1, 2, and 3), a portion or aliquot of each samplewas placed on a clean, inert, glass microscope type slide or plate whichfunctioned as a sample holder in the hyper-spectral imaging and analysissystem. The slide or plate (sample holder) with the sample portion oraliquot was then appropriately positioned and secured (fixed) upon theexamination stage or platform of the hyper-spectral imaging and analysissystem. For each sample type, several separate (individual) dustparticles, of different sizes and shapes, were hyper-spectrally scannedand imaged, for obtaining hyper-spectral images and spectral fingerprint(SFP) or spectral signature pattern types of identification andcharacterization.

Results

At an exemplary characteristic emission wavelength of about 460 nm, the‘average’ emission intensity (in terms of absolute arbitrary units) foreach of the three sample types tested was as follows:

Type 1 samples: dry form dust particles (baseline reference 12,700. orcontrol): Type 2 samples: test suspension of dust particles suspended11,700. in ethanol: Type 3 samples: test suspension of dust particlessuspended 1,600. in ethylene glycol:

Relative to dry form dust particles (as a baseline reference orcontrol), the results show that the ethanol had a relatively very minoreffect (i.e., a decrease of only 1000 units) on reducing the backgroundsignal (average emission intensity) primarily due to the dust particlesin Type 2 samples. By strong contrast, the ethylene glycol had arelatively very major effect (i.e., a decrease of 11,100 units) onreducing the background signal (average emission intensity) primarilydue to the dust particles in Type 3 samples. Based on the results, itwas concluded that for the experimental samples and conditions used andtested in Example 2, ethylene glycol is capable of significantlyreducing background signal (average emission intensity) due to dustparticles which are present in a test suspension of a sample of matter.It was therefore concluded that ethylene glycol is a ‘viable’ andeffective background reducing chemical for use in hyper-spectral imagingand analysis of a sample of matter.

Example 3 Hyper-Spectral Imaging and Analysis of Target-Containing TestSuspensions of {(Dipicolinic Acid [DPA])—Terbium Trichloride [TbCl₃]}Complex, and of Target-Containing Test Suspensions of {Biological AgentBacillus subtilis Spore (Dipicolinic Acid [DPA])—Terbium Trichloride[TbCl₃]} Complex], Suspended in The Background Reducing Chemical beingEthylene Glycol

The rapid and accurate identification of biological agents is a vitaltask for first-responders in order to facilitate timely and appropriateactions in the event of a biological attack. Bacillus anthracis, aspore-forming bacterium and a dangerous pathogen causing the anthraxdisease, is an important example. Among the potential biological warfareagent candidates, Bacillus anthracis spores are of particular concern.First, they are highly resistant to environmental stress and arerelatively easily produced into weapon-grade material outside thelaboratory. Second, anthrax is an infectious disease, requiring medicalattention within 24-48 hours of initial inhalation of more than 104Bacillus anthracis spores. However, the diagnosis of anthrax is notimmediate since it takes 1-60 days for anthrax symptoms to appear inhumans. Therefore, the rapid detection of Bacillus anthracis spores inthe environment prior to infection is an extremely important goal forhuman safety.

Bacillus anthracis bacteria may exist in a spore form. Structurally, aspore consists of a central core surrounded by various protectivelayers. Dipicolinic acid [DPA] is found in these protective layers andaccounts for about 10% of the spore's dry weight. The dipicolinic acid[DPA] can be exploited by being complexed to a chemical type of spectralmarker, for example, terbium trichloride [TbCl₃], which interacts with(i.e., spectrally marks) the spore-forming bacterium Bacillus anthracis(via the dipicolinic acid [DPA] therein) for forming a {biological agentBacillus anthracis spore (dipicolinic acid [DPA])—terbium trichloride[TbCl₃]} complex. The formed complex exhibits a detectable andmeasurable degree or extent of luminescent (i.e., fluorescent or/andphosphorescent) properties, characteristics, and behavior, whenilluminated by different types of electromagnetic radiation or light,such as ultra-violet (UV), visible (VIS), or infrared (IR), types oflight.

In Example 3, the (non-hazardous) biological agent Bacillus subtilis wasused as a functional equivalent substitute for the (extremely hazardous)biological agent Bacillus anthracis. A main objective of Example 3 wasto implement the present invention with respect to: (i) thedetectability of the {(dipicolinic acid [DPA])—terbium trichloride[TbCl₃]} complexes, (ii) the detectability of {biological agent Bacillussubtilis spore (dipicolinic acid [DPA])—terbium trichloride [TbCl₃]}complexes, and (iii) the detectability of {biological agent Bacillussubtilis spore (dipicolinic acid [DPA])—terbium trichloride [TbCl₃]}complexes with the background of native dust particles, and (iv) database creation, identification, and decision making.

Experimental Methods and Procedures

During all measurements the experiments room was completely darkened.

The detectability level was evaluated by three distinct criteria: (1)the area of all pixels expressing the characteristic spectral fingerprint (SFP) of light emitted whether a {(dipicolinic acid [DPA])—terbiumtrichloride [TbCl₃]} complex was exited by a UV light (herein, referredto as Positive Pixels), (2) the fluorescence intensity of every pixelthat expresses the characteristic SFP of {(dipicolinic acid[DPA])—terbium trichloride [TbCl₃]} complexes, and (3) the relationshipbetween SFPs included in Positive Pixels of a given image and spectraldatabase.

(i) Detectability of {(Dipicolinic Acid [DPA])—Terbium Trichloride[TbCl₃]} Complexes

In order to examine the level of detection of {[DPA])—[TbCl₃]} complexesby the hyper-spectral imaging and analysis system, 10 μl (microliters)of DPA and [TbCl₃] solutions (prepared in ethanol) were applied on ablack metal plate, and three spectral images were acquired for each DPAconcentration.

Reagents

[TbCl₃]: 8 mM (millimolar).

DPA: 30 mM, 15 mM, 500 μM (micromolar), 50 μM, 5 μM, 500 nM (nanomolar),50 nM, and 5 nM.

As a control, 10 μl of [TbCl₃], 25 mM applied on a black metal plate wasassayed.

Data Analysis

Hyper-spectral images were analyzed for the characteristic spectrumemitted by the {[DPA])—[TbCl₃]} complexes at a one pixel resolution, andthe total area subtends all positive pixels was calculated for eachhyper-spectral image and DPA concentration. Moreover, the minimum,maximum, and average intensities of the spectral region around thecharacteristic emission peak of the {[DPA])—[TbCl₃]} complex (540±5 nm)were calculated.

Results

The level of detection of {[DPA])—[TbCl₃]} complexes was found toincrease with DPA concentration. Generally, the percentage of pixels(out of all pixels contained in each of the acquired spectral images)expressing the characteristic spectral finger print (SFP) of{[DPA])—[TbCl₃]} complexes (positive pixels) increased with increasingDPA concentration. The highest percentage of positive pixels was foundat a DPA concentration of 30 mM, while the lowest percentage wasachieved at the lowest examined DPA concentration, 5 nM. Additionally,when only [TbCl₃] was assayed, only 7.2% of the pixels were shown to bepositive. The average intensity of the emission peak of the{[DPA])—[TbCl₃]} complex decreased with DPA concentration untilachieving a steady-state around a DPA concentration of 50 μM.

(ii) Detectability of {Biological Agent Bacillus subtilis Spore(Dipicolinic Acid [DPA])—Terbium Trichloride [TbCl₃]} Complexes

The (non-hazardous) biological agent Bacillus subtilis was used as afunctional equivalent substitute for the (extremely hazardous)biological agent Bacillus anthracis.

In order to examine the level of detection of {biological agent Bacillussubtilis spore (dipicolinic acid [DPA])—terbium trichloride [TbCl₃]}complexes, spore suspensions of different concentrations were prepared.Given a stock suspension of Bacillus subtilis spores, five consecutivedilutions were performed. Prior to examining the detectability, sporesof the highest dilution were counted using a standard hemocytometer. Thenumber of spores in each of the dilutions was calculated by multiplyingthe number of spores in the highest dilution by the ratio between thehighest dilution and the dilution in concern. In order to release theDPA molecules from within the spores, the later were broken down byheating at 80° C. for 15 minutes. The heat treated spores were stored ata 4° C. until the spore detectability was examined.

The experimental procedure included the application of 10 μl ofpreheated spore suspension, 10 μl of [TbCl₃] 40 μM solution, and 15 μlof ethylene glycol (background reducing chemical) on a white Teflon®plate. The following final concentrations were achieved: 7.2×10⁶,7.2×10⁵, 7.2×10⁴, 7.2×10³, and 7.2×10² spores/ml (milliliter). Sampleswere examined using the hyper-spectral imaging and analysis system.

Results

The level of detection of complexes comprising of DPA (extracted fromBacillus subtilis spores) and [TbCl₃] molecules was examined. The vastmajority of pixels contained in the acquired spectral images expressedthe characteristic spectral finger print (SFP) of {[DPA])—[TbCl₃]}complexes (positive pixels). Throughout the various sporeconcentrations, the fluorescence intensity of all positive pixels wasfound to decrease with spore concentration. The highest fluorescenceintensity resulted out of the illumination of a sample including 7.2×10⁶while the lowest fluorescence intensity was achieved when a samplecomprising of 7.2×10² spores/ml was illuminated. The results arepresented in FIGS. 3 a and 3 b.

FIG. 3 a is an exemplary empirically determined graphical plot(spectrum) of Emission Intensity (normalized) as a function of EmissionWavelength (nm) of a background-only test suspension [containing onlyobjects of non-interest suspended in the exemplary background reducingchemical being the organic liquid ethylene glycol (monoethylene glycol(MEG)); absent of any object of interest or target (i.e., absent of ahyper-spectrally active target being the exemplary {biological agentBacillus subtilis spore (dipicolinic acid [DPA])—terbium trichloride[TbCl₃]} complex], subjected to hyper-spectral imaging and analysis,showing the spectral fingerprint (SFP) thereof.

FIG. 3 b is an exemplary empirically determined graphical plot(spectrum) of Emission Intensity (normalized) as a function of EmissionWavelength (nm) of a target-containing test suspension [includingobjects of non-interest, and an exemplary object of interest or target(i.e., a hyper-spectrally active target being the exemplary {biologicalagent Bacillus subtilis spore (dipicolinic acid [DPA])—terbiumtrichloride [TbCl₃]} complex, suspended in the exemplary backgroundreducing chemical being the organic liquid ethylene glycol (monoethyleneglycol (MEG))], subjected to hyper-spectral imaging and analysis,showing the spectral fingerprints (SFPs) thereof. In FIG. 3 b, thefollowing reference numbers, in terms of the SFPs are applicable to theindicated concentrations:

SFP 5: 7.2×10² spores/ml.

SFP 4: 7.2×10³ spores/ml.

SFP 2, 3: 7.2×10⁴ spores/ml, 7.2×10⁵, spores/ml.

SFP 1: 7.2×10⁶ spores/ml.

(iii) Detectability of {Biological Agent Bacillus subtilis Spore(Dipicolinic Acid [DPA])—Terbium Trichloride [TbCl₃]} Complexes on(Native) Dust Background

To test the detectability of {biological agent Bacillus subtilis spore(dipicolinic acid [DPA])—terbium trichloride [TbCl₃]} complexes on thebackground of (native) dust, three different spore suspensions wereassayed using the hyper-spectral imaging and analysis system. Since thetotal number of particles to be positioned on the sample plate is 15millions, the number of particles to be spread on the chemical sectionof the sample plate is 3.75 millions. Dust particles ranging between 1and 20 μm in dimension, were suspended in distilled water and using ahemocytometer, spores were counted. 3.75 million dust particles (200 μl(microliters) of dust particles suspension) were applied and evenlyspread on a white Teflon® plate. 10 μl of spore suspension (preheated toachieve the disruption of the spores and the extraction of DPAmolecules) were added to the sample plate. The dust-spore suspension waspartly evaporated by heating the sample with an IR light source, and 10μl of [TbCl₃] 40 mM (final concentration equals 6 mM) and 50 μl ofethylene glycol (as background reducing chemical) was added.

The detectability of the {biological agent Bacillus subtilis spore(dipicolinic acid [DPA])—terbium trichloride [TbCl₃]} complexes on thebackground of native dust was evaluated at 6 different spore suspensionscontaining: 2,500; 5,000; 25,000; 50,000; 250,000 and 2,500,000 spores.Apart from these spore suspensions, a sample containing all reagents butthe spores was assayed as a control. Five hyper-spectral images wereacquired at each spore suspension. That is, conforming to thedetectability requirements of 9000 spores/15,000,000 particles.

Results

The detectability level of complexes comprising of DPA (extracted fromBacillus subtilis spores) and [TbCl₃] molecules on a dusty backgroundwas examined using the hyper-spectral imaging and analysis system. Thepercentage of positive pixels (pixels expressing the characteristicspectral finger print (SFP) of the {biological agent Bacillus subtilisspore (dipicolinic acid [DPA])—terbium trichloride [TbCl₃]} complexes)contained in the acquired hyper-spectral images increased with growingspore count. That is, 12% of the pixels subtending the hyper-spectralimages of the highest spore count (2.5×10⁶ spores) were found to bepositive. On the other hand, only 1.35% of the pixels included inhyper-spectral images taken at a spore count of 2.5×10³ were positive.The percentage of Positive Pixels in the images of the control sample(without spores) was found to be 3.16%. Therefore, the last sporesuspension to result in a Positive Pixel percentage higher than that ofthe control sample is the one yielded from the suspension of 2.5×10⁴spores. The hyper-spectral imaging and analysis system was capable ofdetecting spore suspensions containing at least 25,000 spores.

(iv) Data Base Creation, Identification, and Decision Making

Apart from analyzing the hyper-spectral images for the exact emissionpeak of the {biological agent Bacillus subtilis spore (dipicolinic acid[DPA])—terbium trichloride [TbCl₃]} complex, another approach undertaken was to create large data bases comprising all hyper-spectraincluded in the acquired hyper-spectral images. Following theidentification of the emission peak of interest, all pixels notexpressing the exact emission peak (negative pixels) were discarded fromfurther analyses. Spectral finger prints (SFPs) included within thePositive Pixels were compared against the hyper-spectral data bases as asecond stage validation and a decision was made.

Creating the Background and Target Databases

Two distinct data bases were created. The first corresponds to allavailable spectra of the dusty and particulate background(‘background’), and the other included all spectra corresponding to theBacillus subtilis spores (‘target’). Following the identification of theemission peak, spectral finger prints (SFPs) included within thePositive Pixels were detected, analyzed, grouped, and placed into the‘target’ data base. The ‘target’ data base included all the SFPsdetected in 30 spectral images acquired at six different spore counts.The ‘background’ data base was generated by taking into account all SFPsincluded in the Positive Pixels found in 5 spectral images of dustybackground.

Decision Stage

By comparing the SFPs incorporated in the Positive Pixels of a givenimage to SFPs from the ‘target’ and ‘background’ data bases, PositivePixels were divided into ‘target’ and ‘background’ then correlated. Thisdivision yielded the percentage of Positive Pixels corresponding to‘target’ and the percentage of those matching to ‘background’. Thedecision to be made is based on this pixel distribution, and by so,takes into account the presence of the exact emission peak of the{biological agent Bacillus subtilis spore (dipicolinic acid[DPA])—terbium trichloride [TbCl₃]} complex together with therelationships between the newly acquired SFPs and a large spectral database.

Results

Results of the above described data base creation, identification, anddecision making are presented in FIGS. 4 a and 4 b.

FIG. 4 a is an exemplary empirically determined bar graph of PositivePixels (%) as a function of Spore Count (absolute number) showingdistribution of ‘background’ and ‘target’ spectral fingerprints (SFPs)among Positive Pixels [of the emission peak (540±5 nm) of an exemplaryobject of interest or target (i.e., a hyper-spectrally active targetbeing the exemplary {biological agent Bacillus subtilis spore(dipicolinic acid [DPA])—terbium trichloride [TbCl₃]} complex], as afunction of Spore Count, based on hyper-spectral image data andinformation obtained from FIGS. 3 a and 3 b and similar experiments.

FIG. 4 b is an exemplary empirically determined bar graph of PositivePixels (%) as a function of Spore Count (absolute number) showing‘reproducibility’ of the distribution of ‘background’ and ‘target’spectral fingerprints (SFPs) among Positive Pixels [of the emission peak(540±5 nm) of an exemplary object of interest or target (i.e., ahyper-spectrally active target being the exemplary {biological agentBacillus subtilis spore (dipicolinic acid [DPA])—terbium trichloride[TbCl₃]} complex], as a function of Spore Count, based on hyper-spectralimage data and information obtained by repeating experiments of Example3, as described hereinbelow in Example 4, in accordance with the presentinvention.

For generating the results presented in FIG. 4 b, the detectability ofthe {biological agent Bacillus subtilis spore (dipicolinic acid[DPA])—terbium trichloride [TbCl₃]} complexes on the background ofnative dust was evaluated at 5 different spore suspensions containing:4,500; 9,000; 45,000; 90,000; and 450,000 spores. Apart from these sporesuspensions, a sample containing all reagents but the spores was assayedas a control. Five hyper-spectral images were acquired at each sporesuspension. That is, conforming to the detectability requirements of9000 spores/15,000,000 particles.

Example 4 (Prophetic): Hyper-Spectral Imaging and Analysis ofTarget-Containing Test Solutions or Suspensions of {Biological AgentBacillus subtilis Spore Antigen—Antibody} Complex], Including theBackground Reducing Chemical being Ethylene Glycol Bacillus SubtilisSpores Detection by Immunofluorescence Microscopy-Hyper-Spectral Imagingand Analysis Sample Preparation

Bacillus subtilis spore suspensions (105-106/ml) are fixed in 0.1%glutaraldehyde in PBS for 10 min.

Samples (10 μl) are immediately applied to microscope coverslips (BDH)that are treated with 0.01% (wt/vol) poly-L-lysine (Sigma) (see below).

After 4 min, the liquid is aspirated from the slide, which is thenallowed to dry completely in air at room temperature.

The coverslip is washed in phosphate-buffered saline (PBS) (pH 7.4) andif needed blocked for 15 min with 2% bovine serum albumin (BSA) (SigmaBSA) in PBS at room temperature, and then washed again.

Samples are incubated with 50 μl of staining solution (see below) for 5,10, or 15 minutes.

The samples are rinsed with 10 ml of PBS using gentle flow from a 10 mlpipette.

An aliquot of ethylene glycol is added to the sample as a backgroundreducing chemical.

The coverslips are mounted onto a microscope slide, followed byperforming hyper-spectral imaging and analysis of the samples asdescribed hereinabove.

Polylysine-Coated Coverslips (Polylysine-Coated Microscope Slides,(Sigma))

Sonicate the coverslips (12 mm) in acetone for 15 min at roomtemperature.

Wash the coverslips with distilled water (dH2O) to remove completely theacetone.

Place the coverslips on a clean paper sheet separating each others.Leave the coverslips dry at air.

Gently spread 20 ml polylysine (Sigma) solution (0.1 mg/ml dH2O) on thecoverslips surface.

Leave the polylysine drops on the coverslips dry at room temperature.

Staining Solution:

A 1:1 mix of primary and secondary antibodies in PBS+1% BSA:Antibody dilutions from 1 mg/ml stock solution:1:100/1:500/1:2500/1:12500.

Primary Antibody:

Rabbit purified polyclonal anti Bacillus subtilis/Bacillus cereus spores(USBiological catalog #B0003-27).

Secondary Antibody:

Goat anti rabbit IgG (H+L) ML. The following four options are possible,depending on microscope properties: Cy conjugates are better than FITCor TRITC (slower photobleaching). (Jackson Immunoresearch Laboratories).Cy2 conjugate (A=492/E510 nm) Cat #111-225-144 (Green fluorescence).FITC conjugate (A=492/E=520 nm) Cat #111-095-144 (Green fluorescence).TRITC conjugate (A=550/E=570 nm) Cat #111-025-144 (Red fluorescence).Cy3 conjugate (A=550/E=570 nm) Cat #111-165-144 (Red fluorescence).

The present invention, as illustratively described and exemplifiedhereinabove, has several beneficial and advantageous aspects,characteristics, and features.

First, the present invention is generally applicable for on-line (e.g.,real time or near-real time) or off-line hyper-spectral imaging andanalysis of various different types or kinds of samples of matter,wherein the matter, and at least one object of interest therein, arecomposed or made up of organic or/and inorganic materials or substances,which are in a solid (e.g., particulate) phase, a liquid (e.g., solutionor suspension) phase, or/and a gaseous (e.g., aerosol) phase. Thepresent invention provides the capability of achieving the ‘ultimate’combination of the highly desirable performance parameters of highaccuracy, ‘and’ high precision (high reproducibility), ‘and’ highsensitivity, ‘and’ high resolution, ‘and’ at high speed (short timescale), all at the same time (i.e., simultaneously), be it duringon-line or off-line, in an optimum and highly efficient manner.

Second, the present invention is particularly implementable inapplications involving on-line (real time or near-real time) or off-linehyper-spectral imaging and analysis of a sample of air (i.e., an airsample), for identifying and characterizing an object of interesttherein, wherein the object of interest is a biological agent (e.g., abacterium, a virus, a fungus, or a toxin), or a chemical agent (e.g., anerve agent [e.g., sarin, tabun, or soman], or a chemical poison [e.g.,a cyanide compound, or an organophosphate compound]).

Third, the present invention is particularly implementable inapplications where the sample of air is collected or obtained (e.g., viaa standard type of air sampling or collecting system) from an indoorsource of air (e.g., a post office, an airport, a subway station, ashopping mall, a sports arena, or an office building), or from anoutdoor source of air.

Fourth, the present invention is particularly implementable inapplications wherein the object of interest is a biological agent or achemical agent. More specifically, wherein the sample of matter is asample of air (i.e., an air sample), and the object of interest is abiological agent (e.g., a bacterium, a virus, a fungus, or a toxin), ora chemical agent (e.g., a nerve agent [e.g., sarin, tabun, or soman], ora chemical poison [e.g., a cyanide compound, or a organophosphatecompound]). In general, the object of interest (i.e., biological agentor chemical agent) in the air sample is composed or made up of organicor/and inorganic materials or substances, which are in a solid (e.g.,particulate) phase, a liquid (e.g., solution or suspension) phase,or/and a gaseous (e.g., aerosol) phase. Preferably, the object ofinterest (i.e., biological agent or chemical agent) in the air sample iscomposed or made up of organic or/and inorganic materials or substances,which are in a particulate form solid phase or/and are present (e.g.,absorbed or/and adsorbed) on particles of the air sample. The object ofinterest can be a biological agent, such as the spore-forming bacteriumBacillus anthracis, which is chemically marked (e.g., via terbiumtrichloride [TbCl₃]), or biologically marked (e.g., via antibodies of animmunoassay technique), as part of the main step (procedure) ofpreparing a test solution or suspension of the sample of matter, i.e.,the air sample, for enabling identification and characterization thereofvia hyper-spectral imaging and analysis.

Fifth, the present invention is particularly implementable inessentially any field or area of science and technology involving anapplication which is based on, or involves, the need for on-line (realtime or near-real time) or off-line analyzing a sample of matter, for amain purpose or objective of identifying and characterizing at least oneobject (i.e., entity, material, substance) of interest, usually among avariety of different types or kinds of objects (i.e., entities,materials, substances) of non-interest, in the sample of matter. Suchcharacterization may include determining any number and types or kindsof biological, chemical, or/and physical, properties, characteristics,features, parameters, or/and behavior, of the at least one object ofinterest in the sample of matter.

Based on the preceding, the present invention successfully addresses andovercomes various significant shortcomings and limitations, and widensthe scope, of presently known techniques and methods of hyper-spectralimaging and analysis of a sample of matter. In particular, the presentinvention successfully addresses and overcomes the significantlyproblematic and complicating aspects, and corresponding negative affectsand influences, due to the spatially or/and temporally varying presenceof objects of non-interest (background) in a sample of matter, such as asample of air.

The present invention is readily commercially applicable to a widevariety of different industries.

It is to be fully understood that certain aspects, characteristics, andfeatures, of the invention, which are illustratively described andpresented in the context or format of a plurality of separateembodiments, may also be illustratively described and presented in anysuitable combination or sub-combination in the context or format of asingle embodiment. Conversely, various aspects, characteristics, andfeatures, of the invention, which are illustratively described andpresented in combination or sub-combination in the context or format ofa single embodiment, may also be illustratively described and presentedin the context or format of a plurality of separate embodiments.

Although the invention has been illustratively described and presentedby way of preferred and specific embodiments, and examples thereof, itis evident that many alternatives, modifications, and variations,thereof, will be apparent to those skilled in the art. Accordingly, itis intended that all such alternatives, modifications, and variations,fall within, and are encompassed by, the scope of the appended claims.

All references (patents, patent applications, and publications) cited orreferred to in this specification are herein incorporated in theirentirety by reference into the specification, to the same extent as ifeach individual reference (patent, patent application, or publication)was specifically and individually indicated to be incorporated herein byreference. In addition, citation or identification of any reference inthis specification shall not be construed or understood as an admissionthat such reference represents or corresponds to prior art of thepresent invention.

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1. A method for hyper-spectral imaging and analysis of a sample ofmatter, for identifying and characterizing an object of interesttherein, the method comprising: preparing a test solution or suspensionof the sample of matter, said preparing includes adding to the sample ofmatter a spectral marker specific to the object of interest, such thatif the object of interest is present in said test solution orsuspension, the object of interest when marked with said spectral markerbecomes a hyper-spectrally active target which is hyper-spectrallydetectable and identifiable in said test solution or suspension;generating and collecting hyper-spectral image data and information ofsaid test solution or suspension; and processing and analyzing saidhyper-spectral image data and information, for identifying andcharacterizing said hyper-spectrally active target in said test solutionor suspension, thereby identifying and characterizing the object ofinterest in the sample of matter; wherein said preparing said testsolution or suspension includes adding to said test solution orsuspension a background reducing chemical, wherein said backgroundreducing chemical reduces background interfering effects caused bypresence of objects of non-interest in said test solution or suspension,during the hyper-spectral imaging and analysis, thereby increasinghyper-spectral detectability of said hyper-spectrally active target insaid test solution or suspension.
 2. The method of claim 1, wherein thesample of matter is a sample of air.
 3. The method of claim 1, whereinthe object of interest is a biological agent or a chemical agent.
 4. Themethod of claim 3, wherein said biological agent is selected from thegroup consisting of a bacterium, a virus, a fungus, and a toxin.
 5. Themethod of claim 4, wherein said bacterium is spore-forming bacteriumBacillus anthracis.
 6. The method of claim 3, wherein said chemicalagent is selected from the group consisting of a nerve agent, and achemical poison.
 7. The method of claim 1, wherein said spectral markerspecific to the object of interest is selected from the group consistingof chemical markers and biological markers.
 8. The method of claim 7,wherein said chemical marker is terbium trichloride [TbCl₃].
 9. Themethod of claim 7, wherein said biological marker is an antibody of animmunoassay technique.
 10. The method of claim 1, wherein saidbackground reducing chemical is selected from the group consisting ofsolids, and liquids.
 11. The method of claim 10, wherein said liquid isan organic liquid.
 12. The method of claim 11, wherein said organicliquid is ethylene glycol (monoethylene glycol (MEG) or ethane-1,2-diol)[HOCH₂CH₂OH].
 13. A method for preparing a test solution or suspensionfrom a sample of matter, the test solution or suspension beingparticularly suitable for subjecting to hyper-spectral imaging andanalysis, the method comprising: preparing a solution or suspension ofthe sample of matter, said preparing includes adding to the sample ofmatter a spectral marker specific to an object of interest, such that ifsaid object of interest is present in the test solution or suspension,said object of interest when marked with said spectral marker becomes ahyper-spectrally active target which is hyper-spectrally detectable andidentifiable in the test solution or suspension; wherein said preparingsaid solution or suspension includes adding to said solution orsuspension a background reducing chemical, thereby forming the testsolution or suspension, wherein said background reducing chemicalreduces background interfering effects caused by presence of objects ofnon-interest in the test solution or suspension, during hyper-spectralimaging and analysis of the test solution or suspension, therebyincreasing hyper-spectral detectability of said hyper-spectrally activetarget in the test solution or suspension.
 14. The method of claim 13,wherein the sample of matter is a sample of air.
 15. The method of claim13, wherein said object of interest is a biological agent or a chemicalagent.
 16. The method of claim 15, wherein said biological agent isselected from the group consisting of a bacterium, a virus, a fungus,and a toxin.
 17. The method of claim 16, wherein said bacterium isspore-forming bacterium Bacillus anthracis.
 18. The method of claim 15,wherein said chemical agent is selected from the group consisting of anerve agent, and a chemical poison.
 19. The method of claim 13, whereinsaid spectral marker specific to the object of interest is selected fromthe group consisting of chemical markers and biological markers.
 20. Themethod of claim 19, wherein said chemical marker is terbium trichloride[TbCl₃].
 21. The method of claim 19, wherein said biological marker isan antibody of an immunoassay technique.
 22. The method of claim 13,wherein said background reducing chemical is selected from the groupconsisting of solids, and liquids.
 23. The method of claim 22, whereinsaid liquid is an organic liquid.
 24. The method of claim 23, whereinsaid organic liquid is ethylene glycol (monoethylene glycol (MEG) orethane-1,2-diol) [HOCH₂CH₂OH].
 25. The method of claim 1, wherein saidprocessing and analyzing said hyper-spectral image data and informationincludes evaluating a detectability level of said hyper-spectrallyactive target in said test solution or suspension according to threecriteria: (1) area of all positive pixels in a hyper-spectral imageexpressing characteristic spectral finger prints of light emitted bysaid hyper-spectrally active target, (2) fluorescence intensity of everysaid positive pixel in said hyper-spectral image that expresses saidcharacteristic spectral finger prints of said hyper-spectrally activetarget, and (3) relationship between said characteristic spectral fingerprints included in said positive pixels of said hyper-spectral image andof a hyper-spectral image database.
 26. The method of claim 25, furtherincluding analyzing said hyper-spectral image for said characteristicspectral finger prints at a one pixel resolution, and calculating totalsaid area that subtends all said positive pixels for each saidhyper-spectral image and concentration of said spectral marker.
 27. Themethod of claim 25, further including calculating minimum, maximum, andaverage intensities of a spectral region around a characteristicemission peak of said hyper-spectrally active target.
 28. The method ofclaim 25, further including creating two distinct data bases, wherein afirst data base corresponds to all available spectra of said backgroundinterfering effects, and a second data base includes all spectracorresponding to said hyper-spectrally active target.
 29. The method ofclaim 28, further including comparing spectral finger printsincorporated in said positive pixels of said hyper-spectral image tospectral finger prints from said two distinct data bases, whereby saidpositive pixels are divided into said hyper-spectrally active target andsaid background interfering effects, and then correlating, for yieldingpercentage of said positive pixels corresponding to saidhyper-spectrally active target and percentage of said positive pixelscorresponding to said background interfering effects.
 30. The method ofclaim 29, wherein said percentages of positive pixels are used in a bargraph for showing distribution of said characteristic spectral fingerprints of said hyper-spectrally active target and of said backgroundinterfering effects among said positive pixels of said hyper-spectrallyactive target, as a function of a count of the object of interest.